Phosphoinsitide Regulation of the Golgi

高尔基体的磷酸肌醇调节

• 批准号: 8061658
• 负责人: HELEN L YIN
• 金额: $ 40.32万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-01 至 2013-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8061658
• 关键词: 1-Phosphatidylinositol 4-Kinase; Acyltransferase; Adaptor Signaling Protein; Address; Alzheimer' s Disease; Behavior; Binding; Biology; Capsid Proteins; Cells; Clathrin; Clathrin Adaptors; Cysteine; Data; Dependence; Destinations; Detection; Development; Docking; Early Endosome; Endosomes; Epitopes; Generations; Goals; Golgi Apparatus; Golgi Targeting; Grant; Growth Factor; Guanosine Triphosphate; Guanosine Triphosphate Phosphohydrolases; Heterodimerization; Hybrids; Immunoprecipitation; Integral Membrane Protein; Isotopes; Life; Lipids; Lysosomes; Medial Golgi; Membrane; Membrane Microdomains; Membrane Protein Traffic; Membrane Proteins; Metabolic Diseases; Modeling; Movement; Multivesicular Body; Neurodegenerative Disorders; Organelles; Pathway interactions; Phenotype; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositols; Phosphoric Monoester Hydrolases; Phosphotransferases; Population; Production; Protein Isoforms; Proteins; Proteomics; RNA Interference; Recording of previous events; Recruitment Activity; Recycling; Regulation; Role; Route; Sirolimus; Site; Small Interfering RNA; Sorting - Cell Movement; Testing; Transferase; Transmembrane Transport; Vesicle; adapter protein; base; cellular imaging; density; design; human PHEMX protein; inorganic phosphate; late endosome; membrane activity; mutant; overexpression; palmitoylation; phosphatidylinositol 4-phosphate; protein protein interaction; public health relevance; research study; secretory protein; segregation; tool; trafficking; trans-Golgi Network

DESCRIPTION (provided by applicant): Our overall hypothesis is that PI4KII? (phosphatidylinositol 4 kinase II?), which produces more than 50% of the phosphatidylinositol 4 phosphate (PI4P) in the Golgi, regulates Golgi functions through its localized production of PI4P. The PI4P itself establishes the Golgi's unique organelle identity by recruiting clathrin adapter proteins, such as AP-1, through the coincidence detection of Golgi PI4P and the Arf1 GTPase. In addition, PI4P, as substrate for production of phosphatidylinositol 4, 5 phosphate (PIP2), regulates membrane trafficking from the Golgi. We propose that the localization and enzymatic activity of PI4KII? in the Golgi and Golgi-derived organelles are regulated. Recent data from our lab and others show that the cell has at least two populations of PI4KII? that have different intrinsic catalytic activity and differential association with buoyant vs dense membranes fractions. We recently found that PI4KII?'s catalytic activity, its integral membrane association and partitioning into buoyant noncaveolar "rafts" are critically dependent on its palmitoylation. We therefore propose that these functions of PI4KII? are dynamically regulated by reversible palmitoylation of multiple cysteine residues in PI4KII?. We propose four Specific Aims to test this hypothesis. Aim I. Examine the role of PI4KII? in the generation of dynamic Golgi membrane trafficking carriers that contain PI4KII?. We will characterize these carriers and determine if their generation is dependent on the local synthesis of PI4P per se or downstream synthesis of PIP2. Aim II. Examine the role of PI4P in the recruitment of the AP-3 adaptor protein to endomembranes, to evaluate if it, like the related AP-1, binds target membranes through coincidence detection involving PI4P. Aim III. Determine how palmitoylation regulates PI4KII?. The palmitoylacyl transferase that palmitoylates PI4KII? will be identified and the effect of manipulating its expression on PI4KII? behavior will be examined. Aims I-III focus on the export of membranes from Golgi. Aim IV focuses on the recruitment of PI4KII? to the Golgi. Aim IV. Identify PI4KII?'s primary Golgi targeting motif and interactive proteins. We will identify PI4KII? motifs that are necessary and sufficient to direct PI4KII? to the Golgi prior to palmitoylation and will use an integrated proteomics approach to identify PI4KII? Golgi docking proteins and interactive partners. PUBLIC HEALTH RELEVANCE: Membrane phosphoinositides are major regulators of membrane trafficking, and both their synthesis and degradations are required for dynamic membrane movement and vesicle trafficking within the cell. The experiments in this proposal are designed to examine the role of a lipid kinase that makes an essential lipid in the Golgi. Disruption of this kinase or its misregulation will result in trafficking problems that can contribute to the development of multiple metabolic and neurodegenerative diseases due to improper trafficking of essential components within the cell. Problems with this and other related kinases have already been implicated in neurodegenerative diseases, including Alzheimer's disease.

描述（由申请人提供）：我们的总体假设是PI 4KII？（磷脂酰肌醇4激酶II？），其在高尔基体中产生超过50%的磷脂酰肌醇4磷酸（PI 4P），通过其局部产生PI 4P来调节高尔基体功能。PI 4P本身通过聚集网格蛋白衔接蛋白（如AP-1），通过高尔基体PI 4P和Arf 1 GTdR的同时检测，建立了高尔基体独特的细胞器身份。此外，PI 4P作为生产磷脂酰肌醇4，5磷酸（PIP 2）的底物，调节高尔基体的膜运输。我们建议，本地化和酶活性的PI 4KII？在高尔基体和高尔基体衍生的细胞器中受到调节。我们实验室和其他实验室的最新数据显示，该细胞至少有两个PI 4 KII群体？其具有不同的固有催化活性和与浮力膜级分和致密膜级分的不同缔合。我们最近发现PI 4KII？的催化活性，其整体膜协会和分割成浮力noncaveolar“筏”是严重依赖于其棕榈酰化。因此，我们建议，这些功能的PI 4KII？通过PI 4KII？中多个半胱氨酸残基的可逆棕榈酰化进行动态调节。我们提出了四个具体目标来检验这一假设。艾姆岛研究PI 4KII的作用？在产生含有PI 4KII？的动态高尔基体膜运输载体中。我们将对这些载体进行表征，并确定它们的产生是否依赖于PI 4P本身的局部合成或PIP 2的下游合成。Aim II.检查PI 4P在AP-3衔接蛋白向内膜募集中的作用，以评估其是否与相关AP-1一样通过涉及PI 4P的符合检测结合靶膜。Aim III.确定棕榈酰化如何调节PI 4KII？。棕榈酰化PI 4KII的棕榈酰转移酶？将确定和操纵其表达对PI 4KII？行为将被检查。目的I-III集中于从高尔基体输出膜。目标IV侧重于招募PI 4KII？到高尔基体。目标四。识别PI 4KII？的主要高尔基体靶向基序和相互作用的蛋白质。我们将识别PI 4KII？基序是必要的和足够的指导PI 4KII？高尔基体棕榈酰化之前，并将使用一个综合的蛋白质组学方法来确定PI 4KII？高尔基体对接蛋白和相互作用的伙伴。 公共卫生关系：膜磷酸肌醇是膜运输的主要调节剂，它们的合成和降解都是细胞内动态膜运动和囊泡运输所必需的。在这个建议中的实验旨在研究的作用，使高尔基体中的一种必需的脂质的脂质激酶。这种激酶的破坏或其失调将导致运输问题，这可能有助于多种代谢和神经退行性疾病的发展，由于细胞内的基本组分的不适当运输。这个和其他相关激酶的问题已经涉及神经退行性疾病，包括阿尔茨海默病。