Dynamics of phosphoinositide signaling in cell motility

细胞运动中磷酸肌醇信号传导的动力学

• 批准号: 6743694
• 负责人: JEANNETTE KUNZ
• 金额: $ 26.34万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-08-01 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6743694
• 关键词: SDS polyacrylamide gel electrophoresis; actins; biological signal transduction; cell line; cell motility; chemotaxis; computer program /software; confocal scanning microscopy; double stranded RNA; enzyme activity; growth factor receptors; guanine nucleotide binding protein; immunoprecipitation; lipids; molecular assembly /self assembly; phosphatidylinositols; phospholipids; platelet derived growth factor; polymerase chain reaction; protein biosynthesis; protein localization; protein protein interaction; receptor expression

DESCRIPTION (provided by applicant): Our long-term goal is to define the signaling pathways that control directed cell motility and to understand their roles in pathological processes such as cancer. Cell motility is stimulated by binding of chemotactic stimuli to cell surface receptors and is driven by the dynamic polymerizationn of actin into filaments, a process that is spatially confined to the leading edge. The lipid second messenger phosphatidylinositol-4,5- bisphosphate (PI4,5P2) is recognized as a key signal transducer that links cell surface receptors to the actin cytoskeleton. PI4,5P2 synthesis is essential for actin assembly and cell motility and is locally upregulated at the leading edge in response to PDGF by mechanisms that remain largely obscure. To elucidate these mechanisms we will focus on one particular kinase, type I phosphatidylinositol phosphate kinase alpha (PIPKla), which catalyzes the committed step in PI4,5P2 synthesis. PIPKla localizes to the leading edge of migrating cells and induces actin polymerization in cells stimulated by PDGF. Therefore, PIPKla is a prime candidate for regulating PI4,5P2 synthesis during chemotaxis. Our data indicate that the spatial targeting and activation of PIPKla is tightly regulated by interactions with the PDGF receptor, the small GTPase Rac1, and by binding to the substrate PI4P and the lipid phosphatidic acid (PA). We propose that the coordinate interaction of PIPKla with lipid factors, PDGF receptor and Rac1 results in the specific spatial activation of PI4,5P2 synthesis required for chemotaxis. To test this hypothesis we will combine biochemical and highresolution confocal microscopy approaches in Aim 1 to define the dynamics and mechanisms of PIPKla association with the PDGF receptor complex. Aim 2 will focus on the crosstalk and feedback mechanisms that control the spatial and temporal activation of PIPKla. We will test the hypothesis that Rac1 and PA modulate PIPKla localization and activation. Aim 3 will test the hypothesis that PIPKla mediates PDGF-induced actin assembly and chemotaxis by using inhibitory approaches as well as by mislocalization of PIPKla. Taken together, these experiments will provide novel and detailed information about the spatial and functional relationships between PI4,5P2, the enzymes that produce them, and regulatory components that tightly control PI4,5P2 synthesis. A better understanding of these signaling mechanisms may provide novel therapeutic approaches for the treatment of human diseases.

描述（由申请人提供）：我们的长期目标是定义控制定向细胞运动的信号传导途径，并了解其在癌症等病理过程中的作用。通过趋化刺激与细胞表面受体的结合刺激细胞运动，并由肌动蛋白的动态聚合驱动到细丝中，该过程在空间上局限于前缘。脂质第二信使磷脂酰肌醇-4,5-双磷酸（PI4,5P2）被认为是将细胞表面受体与肌动蛋白细胞骨架联系起来的关键信号传感器。 PI4,5P2合成对于肌动蛋白组装和细胞运动性至关重要，并且在响应PDGF的情况下通过在很大程度上晦涩的机制中局部上调。为了阐明这些机制，我们将重点放在一种特定的激酶I型磷脂酰肌醇磷酸激酶α（PIPKLA）上，该激酶激酶α（PIPKLA）催化PI4,5P2合成中所做的步骤。 PIPKLA定位于迁移细胞的前缘，并在PDGF刺激的细胞中诱导肌动蛋白聚合。因此，Pipkla是调节趋化过程中PI4,5P2合成的主要候选者。我们的数据表明，PIPKLA的空间靶向和激活受到与PDGF受体小型GTPase Rac1的相互作用的严格调节 通过与底物PI4P和脂质磷脂酸（PA）结合。我们提出，PIPKLA与脂质因子，PDGF受体和RAC1的坐标相互作用导致趋化性所需的PI4,5P2合成的特定空间激活。为了检验这一假设，我们将在AIM 1中结合生化和高分辨率共聚焦显微镜方法，以定义PIPKLA与PDGF受体复合物的动力学和机制。 AIM 2将集中在控制PIPKLA的空间和时间激活的串扰和反馈机制上。我们将检验Rac1和PA调节PIPKLA定位和激活的假设。 AIM 3将检验PIPKLA通过使用抑制方法以及通过pipkla错误定位的方法来介导PDGF诱导的肌动蛋白组装和趋化性的假设。综上所述，这些实验将提供有关PI4,5P2（生产它们的酶以及严格控制PI4,5P2合成的调节成分）之间空间和功能关系的新颖和详细信息。更好地了解这些信号传导机制可以为治疗人类疾病提供新的治疗方法。