The Role of PtdIns-5-P in Cell Function and Signaling

PtdIns-5-P 在细胞功能和信号转导中的作用

• 批准号: 6867350
• 负责人: LUCIA RAMEH-PLANT
• 金额: $ 34.11万
• 依托单位: BOSTON BIOMEDICAL RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-06-01 至 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6867350
• 关键词: alcohol phosphotransferase; biological signal transduction; chemical structure function; chimeric proteins; enzyme activity; esterase; lipid biosynthesis; lipid metabolism; phosphatidylinositols; phosphorylation; tissue /cell culture; transfection

DESCRIPTION (provided by applicant): Phosphoinositides are important mediators of hormone and growth factor signals that determine cell proliferation, survival, morphology, metabolism and motility. Phosphoinositide kinases, phosphatases and the proteins that they interact with are directly involved in diseases such as cancer and diabetes. Ptdlns-5-P is the most recently identified phosphoinositide in cells and its function is largely unknown. PIP4k II is a lipid kinase that uses Ptdlns-5-P as a substrate to synthesize Ptdlns-4,5-P2. This phosphoinositide pathway is referred to as the alternative pathway for Ptdlns-4,5-P2 synthesis because it only contributes to a fraction of the total Ptdlns-4,5-P2 in cells. The ultimate goal of this project is to understand the role of Ptdlns-5-P and the alternative pathway for Ptdlns-4,5-P2 synthesis in cell function. Several studies have indicated that the alternative pathway may be regulated by extracellular signals. Further, overexpression of PIP4k II in cells attenuates the signal initiated by insulin by activating the phosphoinositide phosphatase Ship2, while disruption of the PIP4k lib gene in mice increases sensitivity to insulin, a phenotype that resembles Ship2 disruption. The purpose of this study is to test the hypothesis that Ptdlns-5-P is a signaling molecule and that the PIP4k II function in cells is to regulate the levels of Ptdlns-5-P. The first step will be to identify the pathways that lead to Ptdlns-5-P synthesis in cells and to investigate how the levels of this lipid can be regulated by extracellular factors. For this purpose, the enzymes that synthesize Ptdlns-5-P in vitro will be tested for their ability to regulate Ptdlns-5-P in cells. The next step will be to determine which components of the alternative pathway are involved in ship2 activation. PIPk chimeras will be used to determine whether Ptdlns-5-P phosphorylation by PIP4k II is necessary and/or sufficient for Ship2 regulation. The possibility that Ptdlns-5-P is a negative regulator of Ship2 will be explored by testing whether Ptdlns-5-P by itself can regulate Ship2. The third step will be to elucidate the mechanism for Ship2 activation by PIP4k I1. Each domain and regulatory sites in the Ship2 protein will be mutated to assess their role in localization and activation mediated by PIP4k I1.The accomplishment of these goals will set the foundation for designing new strategies to intervene in the disease etiology involving phosphoinositide metabolism malfunction.

描述（由申请人提供）：磷酸肌醇是决定细胞增殖、存活、形态、代谢和运动的激素和生长因子信号的重要介质。磷酸肌醇激酶、磷酸酶及其相互作用的蛋白质直接参与癌症和糖尿病等疾病。 Ptdlns-5-P 是最近在细胞中发现的磷酸肌醇，其功能很大程度上未知。 PIP4k II 是一种脂质激酶，使用 Ptdlns-5-P 作为底物合成 Ptdlns-4,5-P2。该磷酸肌醇途径被称为 Ptdlns-4,5-P2 合成的替代途径，因为它仅占细胞中总 Ptdlns-4,5-P2 的一小部分。该项目的最终目标是了解 Ptdlns-5-P 的作用以及 Ptdlns-4,5-P2 合成在细胞功能中的替代途径。多项研究表明替代途径可能受到细胞外信号的调节。此外，细胞中 PIP4k II 的过度表达会通过激活磷酸肌醇磷酸酶 Ship2 来减弱胰岛素引发的信号，而小鼠中 PIP4k lib 基因的破坏会增加对胰岛素的敏感性，这种表型类似于 Ship2 破坏。本研究的目的是检验以下假设：Ptdlns-5-P 是一种信号分子，PIP4k II 在细胞中的功能是调节 Ptdlns-5-P 的水平。第一步是确定导致细胞中 Ptdlns-5-P 合成的途径，并研究细胞外因素如何调节这种脂质的水平。为此，将测试体外合成 Ptdlns-5-P 的酶在细胞中调节 Ptdlns-5-P 的能力。下一步将确定替代途径的哪些成分参与了 Ship2 的激活。 PIPk 嵌合体将用于确定 PIP4k II 的 Ptdlns-5-P 磷酸化对于 Ship2 监管是否必要和/或充分。通过测试Ptdlns-5-P本身是否能够调节Ship2，探讨Ptdlns-5-P对Ship2负调节的可能性。第三步是阐明 PIP4k I1 激活 Ship2 的机制。 Ship2 蛋白中的每个结构域和调控位点都将发生突变，以评估它们在 PIP4k I1 介导的定位和激活中的作用。这些目标的实现将为设计新策略来干预涉及磷酸肌醇代谢功能障碍的疾病病因学奠定基础。