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: 7099082
• 负责人: JEANNETTE KUNZ
• 金额: $ 5.47万
• 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/7099082
• 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通路，并了解它们在癌症等病理过程中的作用。细胞运动是通过趋化刺激物与细胞表面受体的结合来刺激的，并由肌动蛋白动态聚合成丝来驱动，这一过程在空间上仅限于前缘。脂质第二信使磷脂酰肌醇-4,5-二磷酸 (PI4,5P2) 被认为是连接细胞表面受体与肌动蛋白细胞骨架的关键信号转导器。 PI4、5P2 的合成对于肌动蛋白组装和细胞运动至关重要，并且在响应 PDGF 的前沿局部上调，其机制目前仍不清楚。为了阐明这些机制，我们将重点关注一种特定的激酶，即 I 型磷脂酰肌醇磷酸激酶 α (PIPKla)，它催化 PI4,5P2 合成中的关键步骤。 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 是在趋化过程中调节 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 GTPase Rac1 的相互作用的严格调节， 并通过与底物 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 的协调相互作用导致趋化所需的 PI4、5P2 合成的特定空间激活。为了检验这一假设，我们将在目标 1 中结合生化和高分辨率共聚焦显微镜方法来定义 PIPKla 与 PDGF 受体复合物关联的动力学和机制。目标 2 将重点关注控制 PIPKla 空间和时间激活的串扰和反馈机制。我们将测试 Rac1 和 PA 调节 PIPKla 定位和激活的假设。目标 3 将检验以下假设：PIPKla 通过使用抑制方法以及 PIPKla 的错误定位来介导 PDGF 诱导的肌动蛋白组装和趋化性。总而言之，这些实验将提供有关 PI4,5P2、产生它们的酶以及严格控制 PI4,5P2 合成的调节成分之间的空间和功能关系的新颖而详细的信息。更好地理解这些信号机制可能为治疗人类疾病提供新的治疗方法。