SRC AND RAS PROTO-ONCOGENES AND NGF-INDUCED EVENTS

PC12细胞为研究信号提供了一个有用的模型系统 神经生长因子诱导的信号转导通路 神经元分化。早期的研究表明src和ras相关。 原癌基因家族在神经生长因子刺激的轴突中的关键作用 在PC12细胞中生长。这些项目的主要目标之一是 本申请中提出的是定义Src和RAS家族的角色 调节NGF诱导的轴突形成的早期信号事件中的蛋白质 外延生长。我们将重点关注NGF诱导的三个具体方面 信号转导：i)激活Src家族的激酶，它们与 NGF受体及其在NGF诱导的轴突生长中的作用，2)作用 Shc和Grb2/Sem5在RAS激活中的作用及其调控途径 RAS，以及3)识别参与 MAP激酶和其他细胞事件的激活。初级阶段 这些项目所涉及的策略涉及使用 允许可诱导表达高水平的结构性激活或 显性干扰分子Src、Ras、Shc、Grb2、GAP和Raf，以及 使用亲和纯化策略来识别之前 这些通路的不明成分。另外，还有一秒钟 目标是确定特定相互作用的细胞蛋白质 在Src+的SH3结构域中，Src的变体形式是 在神经元中表达。后一项研究的目标是理解 六个氨基酸是如何插入到该蛋白质的SH3结构域中的 调节其在神经元中的功能活动。

PC12 cells have provided a useful model system to investigate signal transduction pathways involved in nerve growth factor (NGF)-induced neuronal differentiation. Earlier studies have implicated src- and ras family proto-oncogenes as critical components in NGF-stimulated neurite outgrowth in PC12 cells. One of the major objectives of the projects proposed in this application is to define role of Src- and Ras-family proteins in the early signaling events that regulate NGF-induced neurite outgrowth. We will focus on three specific aspects of NGF-induced signaling: i) the activation of Src family kinases, their binding to the NGF receptor, and their role in NGF-induced neurite outgrowth, 2) the role of Shc and Grb2/Sem5 in the activation of Ras and pathways regulated by Ras, and 3) the identification of Ras targets that are involved in the activation of MAP kinases and other cellular events. The primary strategies involved in these projects involve the use of vectors that allow inducible expression of high levels of constitutively activated or dominant-interfering molecules of Src, Ras, Shc, Grb2, GAP, and Raf, and the use of affinity purification strategies to identify previously unidentified components of these pathways. In addition, a second objective, is to identify cellular proteins that specifically interact with the SH3 domain of Src+, the variant form of Src that is specifically expressed in neurons. The goal of these latter studies is to understand how the six amino acid insert within the SH3 domain of this protein regulates its functional activity in neurons.