This thesis describes the work that I have carried out in the Laboratory of Neurophysiolgy at the Université Libre de Bruxelles, under the supervision of Prof. Serge Schiffmann, in collaboration with Prof. Stéphane Schurmans of Université of Liège.The work is divided in two distinct but related projects and the results section is thus divided into two main chapters. The results described are presented in the form of two manuscripts, the first chapter is named “Ciliary phosphoinositides regulation by INPP5E controls Shh signaling by allowing trafficking of Gpr161 in neural stem cells primary cilium”.The second is named “Regulation of phosphoinositides ciliary levels controls trafficking and ciliogenesis in ependymal cells”.Since both manuscripts are comprehensive regarding the results, and methods, these are inserted as such into the thesis.An expanded introduction to the field, placing the results into context, precedes these two chapters. An extended discussion section follows each chapter; it presents some elements of discussion not included in the manuscripts, the implications of the results and the scope for further research. / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

The morbidity of and the mortality from cardiac disease are higher in diabetic patients. Clinical and experimental evidence suggests that diabetes-induced changes at the level of myocardium can, at least partially, contribute to these cardiac problems. The mechanism(s) involved in this diabetic cardiomyopathy is still unclear, but one defect appears to occur in the alpha₁-adrenoceptor system. Altered myocardial sensitivity and responsiveness to alpha₁-adrenoceptor agonists have been reported in experimental diabetes mellitus. Stimulation of alpha₁-adrenoceptors is known to produce a positive inotropic effect and has been recently shown to stimulate the hydrolysis of phosphoinositides. To evaluate the possibility that the changes in the inotropic responsiveness to alpha₁-adrenoceptor stimulation in the diabetic heart could be linked to altered alpha₁-adrenoceptor-stimulated phosphoinositide turnover and further to the development of diabetic cardiomyopathy, we studied contractility and receptor-stimulated phosphoinositide turnover following norepinephrine (in the presence of propranolol) stimulation in right ventricles from male Wistar rats (200-225 g) which were made diabetic with streptozotocin (55 mg/kg, i.v.). Rats were sacrificed six weeks after the induction of diabetes. Diabetic rats were characterized by decreased body weight gain, hypoinsulinemia, hyperglycemia and hyperlipidemia. Stimulation of alpha₁-adrenoceptors by norepinephrine (in the presence of propranolol) in right ventricles resulted in the formation of inositol monophosphate (measured with a radioisotope method) and inositol 1,4,5-trisphosphate (measured with an inositol 1,4,5-trisphosphate protein binding assay kit) in a time- and concentration-dependent manner in both control and diabetic rats. The increase in inositol 1,4,5-trisphosphate levels preceded the increase in the alpha₁-adrenoceptor-mediated positive inotropic effect. Diabetic hearts showed a greater maximum inotropic response to norepinephrine stimulation and also had a higher inositol 1,4,5-trisphosphate levels. However, with the radioisotope method, a decreased inositol monophosphate formation was shown in diabetic hearts compared with controls. Omega-3 fatty acids supplementation (Promega[symbol omitted], 0.5 ml/kg/day) had no significant effect on the changes in norepinephrine-stimulated inositol monophosphate formation in diabetic hearts. In the presence of the cyclooxygenase inhibitor indomethacin or the thromboxane synthetase inhibitor imidazole, the norepinephrine-stimulated positive inotropic effect and inositol 1,4,5-trisphosphate formation were significantly increased in control hearts, but were unaltered in the hearts from diabetics. The addition of the prostacyclin synthetase inhibitor tranylcypromine reduced the norepinephrine-stimulated positive inotropic effect and inositol 1,4,5-trisphosphate formation only in diabetic hearts and had no effect in the controls. While inositol 1,4,5-trisphosphate may be able to mediate only transient inotropic effects produced by alpha₁-adrenoceptor stimulation, diacylglycerol may provoke a sustained positive inotropic effect by activating slow Ca²⁺ channels through stimulation of protein kinase C. Our results showed that the diabetic hearts had a higher protein kinase C activity in the membrane fraction compared with controls and this was accompanied by a decrease in cytosolic protein kinase C activity. The present study suggests that the increases in inositol 1,4,5-trisphosphate levels and the membrane fraction protein kinase C activity may be implicated in the increased inotropic responsiveness to alpha₁-adrenoceptor stimulation in the hearts of the streptozotocin-diabetic rats. The increases in inositol 1,4,5-trisphosphate level and protein kinase C activity could induce Ca²⁺ overload in the diabetic heart which might be involved in the development of diabetic cardiomyopathy. The results from the omega-3 fatty acid study indicate that the changes in cardiac alpha₁-adrenoceptor-mediated inositol phosphates formation cannot contribute to the previously described improved cardiac function of omega-3 fatty acid-treated streptozotocin-diabetic rats. The nature and physiological significance of the enhanced positive inotropic effect and inositol 1,4,5-trisphosphate formation in the control heart with the addition of indomethacin and imidazole is still unclear. The effect of tranylcypromine may indicate the participation of prostaglandins in mediating the enhanced alpha₁-inotropic effect of norepinephrine in the diabetic heart. / Pharmaceutical Sciences, Faculty of / Graduate

The lipid kinase phosphatidylinositol 4-kinase III β (PI4KIIIβ) phosphorylates phosphatidylinositol (PtdIns) to generate PI(4)P in the Golgi. PI4KIIIβ is likely involved in the development of breast cancer as it has been reported genetically amplified in a subset of human breast tumours and is a downstream effector of the eukaryotic elongation factor 1 alpha 2 (eEF1A2), a transforming gene that is amplified and highly expressed in approximately 60% of human breast tumours. The goal of my thesis is to investigate a role for PI4KIIIβ in breast oncogenesis. We show that PI4KIIIβ is highly expressed in approximately 20% of primary human breast tumours. Overexpression of PI4KIIIβ in an invasive breast ductal carcinomas cell line, BT549, increased the production of filopodial actin filament protrusions and enhanced in vitro proliferative capacity. Enhanced PI4KIIIβ expression did not impact the migratory rate of these breast cancer cells. We found that PI4KIIIβ expression activates Akt kinase in the BT549 breast cancer cell line. PI4KIIIβ overexpression led to an increase in the plasma membrane abundance of the PI3K derived PI(3,4,5)P3/PI(3,4)P2 lipids, upstream activators of Akt signalling. PI(4)P and PI(4,5)P2 are precursors to PI(3,4,5)P3 and PI(3,4)P2 generation, however, no changes in the overall cellular abundance or localization of PI(4)P or PI(4,5)P2 were detected in PI4KIIIβ-overexpressing cells. Inhibition of PI4KIIIβ kinase activity, using the drug Pik93, had no effect on PI4KIIIβ-mediated Akt activation. Additionally, ectopic expression of a catalytically inactive PI4KIIIβ also led to increased Akt activity and PI(3,4,5)P3/PI(3,4)P2 plasma membrane abundance. Together, this implies that PI4KIIIβ regulates Akt independently of PI(4)P generation. The PI4KIIIβ interacting protein, Rab11, is likely involved in PI4KIIIβ mediated Akt activation, as RNAi-mediated depletion of Rab11 suppressed the effect of PI4KIIIβ overexpression on Akt activation. Furthermore, PI4KIIIβ overexpression altered cellular Rab11 distribution and led to enhanced recruitment of PI4KIIIβ and Rab11 to recycling endosomes. Therefore, PI4KIIIβ is highly expressed in a subset of breast tumours and upregulated PI4KIIIβ expression enhances filopodia production and cell growth in vitro. Enhanced PI4KIIIβ expression increases PI(3,4,5)P3/PI(3,4)P2 plasma membrane abundance and Akt activation independently of its kinase function, through a mechanism that likely involves Rab11. This work suggests that PI4KIIIβ impacts breast oncogenesis by regulating PI3K/Akt signalling through Rab11 and endosomal trafficking.

The focus of this dissertation is to understand the physicochemical factors that affect the spatiotemporal control of phosphoinositide signaling events. Despite their low abundance in cellular membranes ( ~ 1% of total lipids) phosphoinositides are assuming major roles in the spatiotemporal regulation of cellular signaling, therefore making this group of lipids an attractive area of study, especially for identifying drug targets. The main phosphoinositide studied in this dissertation is phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2], which regulates various intracellular signaling pathways, notably the PI3K/AKT pathway. The PI3K/AKT pathway plays a critical role in regulating diverse cellular functions including metabolism, growth, proliferation, and survival. Thus, dysregulation of the PI3K/AKT pathway is implicated in a number of human diseases including cancer, diabetes, cardiovascular disease and neurological diseases. PI(4,5)P2 regulates phosphoinositide signaling in the PI3K/AKT pathway through interaction of its highly anionic headgroup with polybasic proteins. The highly specific manner that allows hundreds of structurally diverse proteins to interact with lipid species found in such low supply may require the local formation of PI(4,5)P2 clusters (domains). Although a significant amount of evidence has accumulated over the past decade that supports the notion of PI(4,5)P2-rich clusters, our understanding regarding the structural determinants required for cluster formation remains limited. Studies have shown that PI(4,5)P2 clustering is induced by cellular cations interacting with PI(4,5)P2 via electrostatic interactions, suggesting that non-clustering/clustering transitions are particularly sensitive to ionic conditions. However, why some ions are more effectively cluster PI(4,5)P2 than others remains to be understood. For our first research aim, we investigated the effects of divalent (Ca2+) and monovalent cations (Na+, K+ ) on PI(4,5)P2 clustering to understand the ionic environment required for electrostatic PI(4,5)P2 cluster formation. We used monolayers at the air/water interface (Langmuir films) to monitor PI(4,5)P2 molecular packing in the presence of each cation. Our results indicated that Ca2+ individually and Ca2+ along with K+ had a greater effects on PI(4,5)P2 cluster formation than Na+ and K+, individually and combined. We hypothesize that the cations shield the negatively charged headgroups, allowing adjacent PI(4,5)P2 molecules to interact via H- bonding networks. The analysis of the electrostatic environment required for stable PI(4,5)P2 clustering will help us understand important aspects of PI(4,5)P2 mediated signaling events, such as the temporal control of protein binding to PI(4,5)P2 clusters to enhance their function. Another important spatiotemporal modulator that affects the local concentration of PI(4,5)P2 clusters is cholesterol, a steroid present in large quantities (30-40 mole%) in the plasma membrane. Cholesterol has been shown to induce the formation of liquid-ordered domains when interacting with an otherwise gel phase forming lipid, however, the interaction of cholesterol with an inner leaflet lipid species that favors more of a disordered environment to form clusters is poorly understood. We hypothesize that cations along with cholesterol work synergistically to induce PI(4,5)P2 clustering. Thus, our second research aim was to investigate the role of cholesterol on PI(4,5)P2 clustering by monitoring the molecular packing of PI(4,5)P2 in the presence of both cholesterol and cations. This aim was investigated similarly to the first aim with Langmuir trough monolayer film experiments. Our results showed that cholesterol in the presence of Ca2+ had an additive effect leading to the strongest condensation of the monolayer (increase in PI(4,5)P2 packing). Our hypothesis is that Ca2+ significantly reduces the negative electron density of the phosphate groups, allowing the cholesterol hydroxyl group to interact with PI(4,5)P2 headgroup through hydrogen-bond formation. To confirm our hypothesis, we collaborated with a computational group at the NIH that performed all-atom molecular dynamics (MD) simulations that closely agreed with our experimental data. Thus we were able to determine that the cholesterol hydroxyl group directly interacts via hydrogen-bonding with the phosphodiester group as well as the PI(4,5)P2 hydroxyl groups in the 2- and 6-position. The insight into the structural positioning of cholesterol moving closer to the PI(4,5)P2 headgroup region suggests this unique interaction is important for PI(4,5)P2 cluster formation. Other anionic lipid species are suspected to interact with PI(4,5)P2 and strengthen PI(4,5)P2 clustering. We were particularly interested in the interaction of PI(4,5)P2 with phosphatidylinositol (PI) and phosphatidylserine (PS) because both are abundant in the plasma membrane, ~6-10% and ~10-20% respectively, and both electrostatically bind to peripheral proteins. Therefore, the third research aim analyzed the capacity of PI and PS to form stable clusters with PI(4,5)P2. We hypothesize that a mixed PI/PI(4,5)P2 or PS/PI(4,5)P2 domains are ideal for protein binding, since in combination PI or PS with PI(4,5)P2 would provide the necessary negative electrostatic environment, while PI(4,5)P2 would provide the high specificity and additional electrostatics for protein binding. Langmuir trough monolayer films were used to investigate the stabilization of PI/PI(4,5)P2 and PS/PI(4,5)P2 monolayers in the presence of Ca2+. Our results showed a condensation of the monolayer for both PI/PI(4,5)P2 and PS/PI(4,5)P2 with an increase in Ca2+concentrations, which suggests that Ca2+ shields the highly negatively charged phosphomonoester groups of PI(4,5)P2 allowing PI and PS to participate in PI(4,5)P2’s hydrogen-bond network. Interestingly, both PI and PS equally stabilized PI(4,5)P2 cluster formation, therefore it is highly likely that these lipids interact in vivo to form large stable electrostatic domains required for protein binding. The first three aims provided us with information about the physiological relevant environments required for PI(4,5)P2 cluster formation, while the last aim was geared towards understanding the temporal control of protein association with phosphoinositides in the plasma membrane. Specifically, we analyzed the plasma membrane association of PTEN-L, a translation variant protein of PTEN, that has the ability to exit and enter back into cells, unlike classical PTEN. The ability of PTEN-L to facilitate entry across the anionic and hydrophobic layers of the plasma membrane (in the case of direct transport of PTEN-L across the membrane) or into phospholipid transport vesicles (in the case of vesicular transport of PTEN-L across cells) is likely due to the addition of the 173 N-terminal amino acids, the alternative translated region (ATR-domain). Thus, our fourth research aim focused on the biophysical role of the ATR-domain to associate with inner leaflet plasma membrane lipids. Using attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy to monitor secondary structural changes of the ATR-domain upon lipid binding, it was revealed that both PS and PI(4,5)P2 induced conformational change towards a slight increase in β-sheet content in an otherwise unstructured domain suggesting these lipids are required for ATR-domain interaction with the PM. Further studies revealed that the ATR-domain affects the integrity of PS lipid vesicles, further indicating the presence of PS is required to drive ATR-domain across the membrane. This aim provides information on ATR-domain lipid binding preferences aiding in our understanding of the biological and functional role of PTEN-L as a deliverable tumor suppressor protein. The overall goal of the research in this dissertation is to understand factors that fine-tune PI(4,5)P2 cluster formation in space and time. Our first three research aims were designed to understand the synergistic effects of spatiotemporal modulators (cations, cholesterol, and anionic lipids) on local concentration of PI(4,5)P2 clusters. Our results indicate that Ca2+, cholesterol, and the presence of anionic lipids PI and PS all induce stable domains, thus it is highly likely this is part of the biological environment required in vivo for cationic proteins to bind. The last aim, the association of the ATR-domain with phospholipids in the plasma membrane, provided evidence that PS is likely required to drive the ATR-domain across the plasma membrane. This dissertation unifies nearly two decades worth of research by shedding light on synergistic modulators of PI(4,5)P2 cluster formation (Figure 1). Thus, this work has potentially far reaching consequences for understanding temporal control of the spatially resolved protein activity.

The focus of this dissertation is to understand the physicochemical factors that affect the spatiotemporal control of phosphoinositide signaling events. Despite their low abundance in cellular membranes ( ~ 1% of total lipids) phosphoinositides are assuming major roles in the spatiotemporal regulation of cellular signaling, therefore making this group of lipids an attractive area of study, especially for identifying drug targets. The main phosphoinositide studied in this dissertation is phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2], which regulates various intracellular signaling pathways, notably the PI3K/AKT pathway. The PI3K/AKT pathway plays a critical role in regulating diverse cellular functions including metabolism, growth, proliferation, and survival. Thus, dysregulation of the PI3K/AKT pathway is implicated in a number of human diseases including cancer, diabetes, cardiovascular disease and neurological diseases. PI(4,5)P2 regulates phosphoinositide signaling in the PI3K/AKT pathway through interaction of its highly anionic headgroup with polybasic proteins. The highly specific manner that allows hundreds of structurally diverse proteins to interact with lipid species found in such low supply may require the local formation of PI(4,5)P2 clusters (domains). Although a significant amount of evidence has accumulated over the past decade that supports the notion of PI(4,5)P2-rich clusters, our understanding regarding the structural determinants required for cluster formation remains limited. Studies have shown that PI(4,5)P2 clustering is induced by cellular cations interacting with PI(4,5)P2 via electrostatic interactions, suggesting that non-clustering/clustering transitions are particularly sensitive to ionic conditions. However, why some ions are more effectively cluster PI(4,5)P2 than others remains to be understood. For our first research aim, we investigated the effects of divalent (Ca2+) and monovalent cations (Na+, K+ ) on PI(4,5)P2 clustering to understand the ionic environment required for electrostatic PI(4,5)P2 cluster formation. We used monolayers at the air/water interface (Langmuir films) to monitor PI(4,5)P2 molecular packing in the presence of each cation. Our results indicated that Ca2+ individually and Ca2+ along with K+ had a greater effects on PI(4,5)P2 cluster formation than Na+ and K+, individually and combined. We hypothesize that the cations shield the negatively charged headgroups, allowing adjacent PI(4,5)P2 molecules to interact via H- bonding networks. The analysis of the electrostatic environment required for stable PI(4,5)P2 clustering will help us understand important aspects of PI(4,5)P2 mediated signaling events, such as the temporal control of protein binding to PI(4,5)P2 clusters to enhance their function. Another important spatiotemporal modulator that affects the local concentration of PI(4,5)P2 clusters is cholesterol, a steroid present in large quantities (30-40 mole%) in the plasma membrane. Cholesterol has been shown to induce the formation of liquid-ordered domains when interacting with an otherwise gel phase forming lipid, however, the interaction of cholesterol with an inner leaflet lipid species that favors more of a disordered environment to form clusters is poorly understood. We hypothesize that cations along with cholesterol work synergistically to induce PI(4,5)P2 clustering. Thus, our second research aim was to investigate the role of cholesterol on PI(4,5)P2 clustering by monitoring the molecular packing of PI(4,5)P2 in the presence of both cholesterol and cations. This aim was investigated similarly to the first aim with Langmuir trough monolayer film experiments. Our results showed that cholesterol in the presence of Ca2+ had an additive effect leading to the strongest condensation of the monolayer (increase in PI(4,5)P2 packing). Our hypothesis is that Ca2+ significantly reduces the negative electron density of the phosphate groups, allowing the cholesterol hydroxyl group to interact with PI(4,5)P2 headgroup through hydrogen-bond formation. To confirm our hypothesis, we collaborated with a computational group at the NIH that performed all-atom molecular dynamics (MD) simulations that closely agreed with our experimental data. Thus we were able to determine that the cholesterol hydroxyl group directly interacts via hydrogen-bonding with the phosphodiester group as well as the PI(4,5)P2 hydroxyl groups in the 2- and 6-position. The insight into the structural positioning of cholesterol moving closer to the PI(4,5)P2 headgroup region suggests this unique interaction is important for PI(4,5)P2 cluster formation. Other anionic lipid species are suspected to interact with PI(4,5)P2 and strengthen PI(4,5)P2 clustering. We were particularly interested in the interaction of PI(4,5)P2 with phosphatidylinositol (PI) and phosphatidylserine (PS) because both are abundant in the plasma membrane, ~6-10% and ~10-20% respectively, and both electrostatically bind to peripheral proteins. Therefore, the third research aim analyzed the capacity of PI and PS to form stable clusters with PI(4,5)P2. We hypothesize that a mixed PI/PI(4,5)P2 or PS/PI(4,5)P2 domains are ideal for protein binding, since in combination PI or PS with PI(4,5)P2 would provide the necessary negative electrostatic environment, while PI(4,5)P2 would provide the high specificity and additional electrostatics for protein binding. Langmuir trough monolayer films were used to investigate the stabilization of PI/PI(4,5)P2 and PS/PI(4,5)P2 monolayers in the presence of Ca2+. Our results showed a condensation of the monolayer for both PI/PI(4,5)P2 and PS/PI(4,5)P2 with an increase in Ca2+concentrations, which suggests that Ca2+ shields the highly negatively charged phosphomonoester groups of PI(4,5)P2 allowing PI and PS to participate in PI(4,5)P2’s hydrogen-bond network. Interestingly, both PI and PS equally stabilized PI(4,5)P2 cluster formation, therefore it is highly likely that these lipids interact in vivo to form large stable electrostatic domains required for protein binding. The first three aims provided us with information about the physiological relevant environments required for PI(4,5)P2 cluster formation, while the last aim was geared towards understanding the temporal control of protein association with phosphoinositides in the plasma membrane. Specifically, we analyzed the plasma membrane association of PTEN-L, a translation variant protein of PTEN, that has the ability to exit and enter back into cells, unlike classical PTEN. The ability of PTEN-L to facilitate entry across the anionic and hydrophobic layers of the plasma membrane (in the case of direct transport of PTEN-L across the membrane) or into phospholipid transport vesicles (in the case of vesicular transport of PTEN-L across cells) is likely due to the addition of the 173 N-terminal amino acids, the alternative translated region (ATR-domain). Thus, our fourth research aim focused on the biophysical role of the ATR-domain to associate with inner leaflet plasma membrane lipids. Using attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy to monitor secondary structural changes of the ATR-domain upon lipid binding, it was revealed that both PS and PI(4,5)P2 induced conformational change towards a slight increase in β-sheet content in an otherwise unstructured domain suggesting these lipids are required for ATR-domain interaction with the PM. Further studies revealed that the ATR-domain affects the integrity of PS lipid vesicles, further indicating the presence of PS is required to drive ATR-domain across the membrane. This aim provides information on ATR-domain lipid binding preferences aiding in our understanding of the biological and functional role of PTEN-L as a deliverable tumor suppressor protein. The overall goal of the research in this dissertation is to understand factors that fine-tune PI(4,5)P2 cluster formation in space and time. Our first three research aims were designed to understand the synergistic effects of spatiotemporal modulators (cations, cholesterol, and anionic lipids) on local concentration of PI(4,5)P2 clusters. Our results indicate that Ca2+, cholesterol, and the presence of anionic lipids PI and PS all induce stable domains, thus it is highly likely this is part of the biological environment required in vivo for cationic proteins to bind. The last aim, the association of the ATR-domain with phospholipids in the plasma membrane, provided evidence that PS is likely required to drive the ATR-domain across the plasma membrane. This dissertation unifies nearly two decades worth of research by shedding light on synergistic modulators of PI(4,5)P2 cluster formation (Figure 1). Thus, this work has potentially far reaching consequences for understanding temporal control of the spatially resolved protein activity.

Metastatic cancer is more dangerous and difficult to treat than pre-metastatic cancer. Ninety percent of cancer-related deaths are caused by metastatic cancer. When cells go through metastases, they go through changes that allow them to break away from the primary tumor and invade secondary tissues. These changes, in lipid membrane composition and cellular glycocalyx, make the cell more resistant to therapeutics. Actin cytoskeleton contractility plays a major role in these changes, as increased contractility has been linked to upregulation of phosphoinositides and production of glycoproteins. Light induced molecular adsorption of proteins (LIMAP) was used to control the actin arrangement and cell shape in order to mimic and study metastatic cells. Negatively charged proteins electrostatically adhere to the surface in order to create patterns for the cells to stick. Neutravidin was conjugated to poly(glutamic acid) to improve attachment to the surface. We observed differences in cell shape and phosphoinositide behavior based on LIMAP patterning. Additionally, expression of key glycoproteins related to cancer metastasis increased with increased actin contractility. The actin cytoskeleton was the main driver of changes to the cell membrane and glycocalyx.

Cells transduce signals from the external environment to the inside through phosphatidylinositol-3,4,5-phosphate (PIP3), a major signaling lipid on the plasma membrane. PIP3 is generated by the action of a family of lipid kinases called Class I phosphoinositide 3-kinases (PI3Ks) and controls an array of essential cellular functions including growth, proliferation, survival, metabolism and cytoskeletal architecture. PI3Ks are large heterodimeric complexes composed of a catalytic p110 subunit and a regulatory subunit. Crucial to healthy PIP3 production is the interpretation of diverse activating inputs arising from signaling proteins on the membrane by these subunits. A member of the PI3K family, PI3Kγ is a master regulator of immune functions with therapeutic implications in cancer immunity and inflammatory disease. PI3Kγ is distinct from other well studied PI3Ks due to the presence of unique regulatory mechanisms that control its ability to integrate signals from G-protein coupled receptors, small GTPases, immunoglobulin receptors and toll-like receptors. However, unlike the other well characterized PI3Ks, there are significant gaps in understanding of the molecular details of these mechanisms and how regulatory processes are translated into functions elicited by PI3Kγ in its unique milieu within the immune system. To understand PI3Kγ regulation, I utilized a synergy of cutting-edge approaches including protein biochemistry, X-ray crystallography, cryo-electron microscopy and hydrogen-deuterium exchange mass spectrometry to elucidate the unique regulatory features within its catalytic and regulatory subunits and how these features are disrupted in disease. These studies significantly advanced our understanding of how this enzyme functions and provided novel avenues for potentially targeting the enzyme better in therapy. This dissertation will consist of an introduction chapter summarizing PI3Kγ regulation and its role in disease, followed by three data chapters investigating previously uncharacterized regulatory mechanisms that control its function and how these mechanisms are implicated in disease. These data chapters are followed by a final chapter describing conclusions and future directions. In summary, the work presented in this thesis provides novel insights into the unique regulatory features in the catalytic and regulatory subunits of PI3Kγ that mediate its stimulation by upstream activating partners and the mechanisms by which these features are disrupted in disease. Further, these studies have facilitated the effective characterization of potent molecules that can specifically target PI3Kγ in disease. Altogether, the findings of this dissertation constitute a major advancement in our understanding of PI3K regulation. / Graduate

Yes / Background: The receptor for activated C-kinase 1 (RACK1) is a conserved protein belonging to the WD40 repeat family of proteins. It folds into a beta propeller with seven blades which allow interactions with many proteins. Thus it can serve as a scaffolding protein and have roles in several cellular processes. Results: We identified the product of the Dictyostelium discoideum gpbB gene as the Dictyostelium RACK1 homolog. The protein is mainly cytosolic but can also associate with cellular membranes. DdRACK1 binds to phosphoinositides (PIPs) in protein-lipid overlay and liposome-binding assays. The basis of this activity resides in a basic region located in the extended loop between blades 6 and 7 as revealed by mutational analysis. Similar to RACK1 proteins from other organisms DdRACK1 interacts with G protein subunits alpha, beta and gamma as shown by yeast two-hybrid, pulldown, and immunoprecipitation assays. Unlike the Saccharomyces cerevisiae and Cryptococcus neoformans RACK1 proteins it does not appear to take over Gβ function in D. discoideum as developmental and other defects were not rescued in Gβ null mutants overexpressing GFP-DdRACK1. Overexpression of GFP-tagged DdRACK1 and a mutant version (DdRACK1mut) which carried a charge-reversal mutation in the basic region in wild type cells led to changes during growth and development. Conclusion: DdRACK1 interacts with heterotrimeric G proteins and can through these interactions impact on processes specifically regulated by these proteins. / This work was supported by the DFG and SFB670. TYR acknowledges support from the Professorinnen Program of the University of Cologne.

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