REGULATION OF AXON GUIDANCE BY ENA/VASP PROTEINS

ENA/VASP 蛋白对轴突引导的调节

• 批准号: 6583536
• 负责人: Erik W Dent
• 金额: $ 4.16万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-12-01 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6583536
• 关键词: A kinase anchoring protein; biological signal transduction; brain derived neurotrophic factor; fluorescence resonance energy transfer; genetically modified animals; growth cones; immunoprecipitation; laboratory mouse; membrane proteins; molecular cloning; nerve /myelin protein; neuronal guidance; neuroregulation; phosphorylation; postdoctoral investigator; protein protein interaction; protein structure function

DESCRIPTION (provided by applicant): The overall goal of this application is to elucidate the function of Ena/VASP phosphorylation by protein kinase A (PKA) in axon outgrowth and guidance in the developing central nervous system. First, Ena/VASP-null mice will be generated. If it is not possible to create triple knock-out mice due to early prenatal lethality, a conditional knock-out strategy will be employed by means of the Cre/lox system of recombination. Second, phosphorylation mutants of Ena/VASP proteins will be introduced into Ena/VASP-null neurons and their effects on growth cone motility and axon outgrowth will be assessed. To test the function of Ena/VASP phosphorylation on axon guidance cortical neurons containing phosphorylation mutants will be exposed to gradients of netrin and BDNF; two molecules known to signal through PKA. Ena/VASP proteins have also been implicated in binding A kinase anchoring proteins (AKAPs). In order to determine which AKAPs associate with Ena/VASP proteins in cortical neurons co-immunoprecipitations and gel overlays will be performed with the type II regulatory subunit of PKA. AKAPs discovered to bind Ena/VASP proteins will be cloned and labeled with YFP. Fluorescent resonance energy transfer (FRET) will be performed with CFP-Ena/VASP proteins to determine where and when Ena/VASP proteins and AKAPs interact in growth cones. An elucidation of the function of Ena/VASP proteins in CNS development will be important for understanding human CNS diseases where directed neuronal migration and outgrowth are impaired.

描述(申请人提供)：本申请的总体目标是阐明蛋白激酶A(PKA)的Ena/Vasp磷酸化在轴突生长和在发育中的中枢神经系统中的指导作用。首先，将产生Ena/Vasp缺失的小鼠。如果由于早期的产前死亡而不可能产生三重基因敲除小鼠，则将通过重组的Cre/lox系统来采用条件基因敲除策略。其次，将Ena/Vasp蛋白的磷酸化突变体引入Ena/Vasp缺失神经元，并评估它们对生长锥运动和轴突生长的影响。为了测试Ena/Vasp在轴突导向上的磷酸化功能，含有磷酸化突变体的皮质神经元将暴露于Netrin和BDNF的梯度中，这两个分子通过PKA发出信号。Ena/Vasp蛋白也与结合A激酶锚定蛋白(AKAP)有关。为了确定哪些AKAP与皮层神经元中的ena/Vasp蛋白相关联，将与PKA的II型调节亚基进行免疫共沉淀和凝胶覆盖。已发现与Ena/Vasp蛋白结合的AKAP将被克隆并用YFP标记。荧光共振能量转移(FRET)将与CFP-EnA/Vasp蛋白一起进行，以确定EnA/Vasp蛋白和AKAP在生长锥体中何时何地相互作用。阐明Ena/Vasp蛋白在中枢神经系统发育中的功能对于了解人类中枢神经系统疾病的定向迁移和突起生长受损具有重要意义。