CONSEQUENCES OF FAK DISRUPTION ON NEURONAL DEVELOPMENT

FAK 破坏对神经元发育的影响

• 批准号: 6136418
• 负责人: HILARY E BEGGS
• 金额: $ 3.92万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-02-07 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6136418
• 关键词: adenosine triphosphate; binding sites; developmental neurobiology; enzyme activity; focal adhesion kinase; gene targeting; genetically modified animals; growth cones; laboratory mouse; point mutation

DESCRIPTION(Applicant's abstract reproduced verbatim): Tight regulation of the focal adhesion kinase (FAK) tyrosine kinase activity is likely essential for normal growth and motility of developing neurons. FAK has 3 alternatively spliced isoforms that specifically regulate kinase activity in the brain, suggesting the importance of exquisite regulation of FAK in the nervous system. Cell culture studies have demonstrated that FAK can mediate integrin-stimulated cell migration and attachment to the cytoskeleton, activate the MAP kinase pathway, trigger anchorage-dependent survival signals, and respond to a variety of growth factors. Given these abundant and biologically significant functions, an essential challenge is to clarify the role of specific FAK signaling pathways in an in vivo, physiological context. Germline FAK deficiency results in early lethality (E8.5) precludes later developmental study. Therefore, in this fellowship I will: Aim 1) generate a "knock-in" point mutation in the ATP binding site of FAK which will result in a kinase dead (KD) FAK protein. The hypothesis is that a kinase-dead FAK mouse phenotype will be less severe than a complete null and reveal those physiological processes which are reliant on kinase signaling pathways and those which are kinase-independent. Aim 2) Characterize the in vivo consequences of kinase-dead (KD) FAK in axonal tract formation and cell survival. Assay growth cone navigation errors at the mouse optic chiasm and evaluate neuronal cell survival in the neural crest and the dorsal root ganglia (DRG). Aim 3) Evaluate the role of KD-FAK in regulation of integrin-mediated contacts, phosphorylation of proteins associated with the neuronal cytoskeleton, response to growth factor stimulation, and in the induction of anchorage-independent cell survival.

描述（逐字复制申请人的摘要）：严格监管 粘着斑激酶 (FAK) 酪氨酸激酶活性可能对于 发育中神经元的正常生长和运动。 FAK有3个可选 特异性调节大脑中激酶活性的剪接亚型， 表明 FAK 在神经系统中精细调节的重要性。 细胞培养研究表明，FAK 可以介导整合素刺激 细胞迁移和附着于细胞骨架，激活 MAP 激酶 途径，触发锚定依赖性生存信号，并对各种 的生长因子。鉴于这些丰富且具有生物学意义的功能， 一个重要的挑战是阐明特定 FAK 信号传导的作用 体内生理环境中的途径。种系 FAK 缺乏结果 早期致死率（E8.5）排除了以后的发育研究。因此，在 在这次奖学金中我将： 目标1）在FAK的ATP结合位点产生“敲入”点突变 这将导致激酶死亡 (KD) FAK 蛋白。假设是一个 激酶死亡的 FAK 小鼠表型不会比完全无效的小鼠严重 揭示那些依赖于激酶信号传导的生理过程 途径和不依赖于激酶的途径。 目标 2) 表征轴突中激酶死亡 (KD) FAK 的体内后果 束形成和细胞存活。测定生长锥导航误差 小鼠视交叉并评估神经嵴中神经元细胞的存活率 背根神经节（DRG）。 目标 3) 评估 KD-FAK 在整合素介导的接触调节中的作用， 与神经元细胞骨架相关的蛋白质的磷酸化，反应 生长因子刺激，以及诱导不依赖锚定的 细胞存活。