京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13386号 / 論医博第2218号 / 新制||医||1048(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 椛島 健治, 教授 妻木 範行, 教授 安達 泰治 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18886号 / 医博第3997号 / 新制||医||1009(附属図書館) / 31837 / 京都大学大学院医学研究科医学専攻 / (主査)教授 三森 経世, 教授 戸口田 淳也, 教授 開 祐司 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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.

Lithium, a drug used for the treatment of bipolar disorder, has been shown to affect different aspects of neuronal development such as neuritogenesis, neurogenesis and survival. The underlying mechanism responsible for lithium's influence on neuronal development, however, still remains to be elucidated. In the present study, we demonstrate that lithium increases the phosphorylation of extracellular-signal regulated kinases (ERKs) and protein kinase B (Akt) and promotes neurite outgrowth in mouse N2a neuroblastoma cells (N2a). The inactivation of mitogen-activated protein kinase kinase (MEK)/ERKs signaling with a MEK inhibitor inhibits neurite outgrowth, but it enhances Akt activation in lithium-treated N2a cells. Furthermore, the inactivation of phosphoinositide-3-kinase (PI3K)/Akt signaling with a PI3K inhibitor increases both lithium-induced ERKs activation and lithium-induced neurite outgrowth. Taken together, our study suggests that lithium-induced neurite outgrowth in N2a cells is regulated by cross-talk between the MEK/ERKs and PI3K/Akt pathways and requires the activation of the MEK/ERKs signaling.

A role for the PI3K/Akt/mTOR pathway in cardiac hypertrophy has been well documented. We reported that NFκB activation is needed for cardiac hypertrophy in vivo. To investigate whether both NFκB activation and PI3K/Akt/mTOR signaling participate in the development of cardiac hypertrophy, two models of cardiac hypertrophy, namely, induction in caAkt-transgenic mice and by aortic banding in mice, were employed. Rapamycin (2 mg/kg/daily), an inhibitor of the mammalian target of rapamycin, and the antioxidant pyrrolidine dithiocarbamate (PDTC; 120 mg/kg/daily), which can inhibit NFκB activation, were administered to caAkt mice at 8 weeks of age for 2 weeks. Both rapamycin and PDTC were also administered to the mice immediately after aortic banding for 2 weeks. Administration of either rapamycin or PDTC separately or together to caAkt mice reduced the ratio of heart weight/body weight by 21.54, 32.68, and 42.07% compared with untreated caAkt mice. PDTC administration significantly reduced cardiac NFκB activation by 46.67% and rapamycin significantly decreased the levels of p70S6K by 34.20% compared with untreated caAkt mice. Similar results were observed in aortic-banding-induced cardiac hypertrophy in mice. Our results suggest that both NFκB activation and the PI3K/Akt signaling pathway participate in the development of cardiac hypertrophy in vivo.

As the primary antagonist to the tumorigenic PI3K/AKT pathway, PTEN is classified as a tumor suppressor. The inactivation of PTEN through genetic or post-translational modifications is a critical step in the tumorigenesis of many breast cancers (BCs). SIPL1 is a novel protein which was identified as a PTEN negative regulator. To further explore SIPL1-mediated PTEN inactivation, we analyzed 17 datasets covering 3484 BC cases and 228 normal individuals. SIPL1 gene amplification and increased mRNA expression correlates with the progression and poor prognosis of ER and/or PR positive tumors. Furthermore, examination of a BC tissue microarray containing 224 tumor cases revealed elevated SIPL1 protein expression in ER+ and PR+ tumors and was associated with greater AKT activation. Additionally, ectopic expression of SIPL1 in CHO-K1 cells resulted in increased AKT activation and cell proliferation, and cytoskeleton reorganization alongside with PTEN downregulation. SIPL1 contributes to the linear polyubiquitination of NEMO, suggesting a role for SIPL1 in PTEN ubiquitination. Indeed, it was SIPL1, not the SIPL1-∆UBL (a PTEN-binding defective mutant) which robustly induced PTEN polyubiquitination in a lysine (K) 63-dependent but K48-independent manner. While K48-linked polyubiquitin chains direct protein degradation, K63-linked chains regulate a variety of protein functions. SIPL1 binds polyubiquitinated PTEN with significantly higher affinity than non-ubiquitinated PTEN. A SIPL1 mutant, SIPL1-TFLV, is unable to cause PTEN ubiquitination but is capable of PTEN association. Collectively, our results reveal that SIPL1 interacts with PTEN with a low affinity, which results in PTEN polyubiquitination, and that the modification may stabilize the association between SIPL1 and PTEN. We propose a model where SIPL1 mediates the K63-linked ubiquitination of PTEN inactivating it. The downregulation of PTEN, when paired with the tumor-promoting effects of ER and/or PR, stimulates breast tumorigenesis. SIPL1 is an important BC marker and future research should focus on its potential as a therapeutic target. / Thesis / Doctor of Philosophy (PhD)

No description available.

In this study, we investigated differential activation and the role of two mTOR complexes in cell migration of prostate cancer cells. Specific knock-down of endogenous RAPTOR and RICTOR by siRNA resulted in decreased cell migration in LNCaP, DU145, and PC3 cells indicating that both mTORC1 and mTORC2 are required for cell migration. EGF treatment induced the activation of both mTORC1 and mTORC2 as determined by complex-specific phosphorylation of mTOR protein. Specific knock-down or inhibition of Rac1 activity in PC3 cells blocked EGF-induced activation of mTORC2, but had no effect on mTORC1 activation. Furthermore, the over-expression of constitutively active Rac1 (Rac1Q61L) resulted in significant increase in cell migration and activation of mTORC2 in PC3 cells, but had no effect on mTORC1 activation. Constitutively active Rac1 (Rac1Q61L) in PC3 cells was localized in the plasma membrane and was found to be in a protein complex which contained mTOR and RICTOR proteins, but not RAPTOR. In conclusion, we suggested that EGF-induced activation of Rac1 causes the phosphorylation/activation of mTORC2 via RICTOR, specific regulator of mTORC2 activation in numerous cancer cells. The major role played by mTOR in a wide array of cancers has in the recent decades led to the development of numerous mTOR inhibitors. One of the drawback of these first generation mTOR inhibitors are that m TORC1 activity is inhibited but effect on mTORC2 activity require high dosages and prolonged exposure in different cancer cell types including HeLa, PC3, LNCaP, and A549. High dosage of rapamycin and its associated rapalogs required for mTORC2 inhibition is clinically unsuitable. Studies have shown that the dual mTORC1/C2 inhibitors trigger feedback loops causing metastasis and affect the cell viability of normal tissues in vitro and in vivo. There is a need for specific mTORC1 and mTORC2 inhibitor, which overcome the disadvantages of the previously developed mTOR inhibitors. The Rac1-RICTOR axis suggested in this study could be used as a potential target for the development of mTORC2 inhibitor and lead to a potential therapeutic treatment for aggressive prostate cancer.