CONSEQUENCES OF FAK DISRUPTION ON NEURONAL DEVELOPMENT

描述（申请人的摘要逐字复制）： 粘着斑激酶（FAK）酪氨酸激酶活性可能是 发育中的神经元的正常生长和运动。FAK有3个可选 特异性调节脑中激酶活性的剪接异构体， 提示FAK在神经系统中精细调节的重要性。 细胞培养研究已经证明FAK可以介导整合素刺激的 细胞迁移和附着于细胞骨架，激活MAP激酶 通路，触发锚定依赖性生存信号，并对各种 的生长因子。鉴于这些丰富的和生物学上重要的功能， 一个重要的挑战是阐明特异性FAK信号传导的作用 在体内生理环境中的途径。生殖系FAK缺陷结果 早期致死率（E8.5）排除了以后的发育研究。因此在 我将在此团契中： 目的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， 激酶死亡FAK小鼠表型将不如完全无效的严重， 揭示那些依赖于激酶信号传导的生理过程 和那些不依赖激酶的途径。 目的2）研究激酶死亡（KD）的FAK在轴突中的作用。 束形成和细胞存活。试验生长锥导航错误 小鼠视交叉和评估神经嵴中的神经元细胞存活， 背根神经节（DRG）。 目的3）研究KD-FAK在整合素介导的接触中的调节作用， 与神经元细胞骨架相关的蛋白质的磷酸化， 生长因子刺激，并在诱导锚定非依赖性 细胞存活

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.