SIGNAL TRANSDUCTION PATHWAYS FOR NEURONAL MIGRATION

描述：该提案旨在识别和表征神经元迁移和皮质发育所需的信号通路的组成部分。虽然Dbcn、mDab1和βLis1基因的缺陷都会导致严重的皮质异常，但对这些基因产物参与的信号通路知之甚少。尤其是沃尔什和他的同事们今年早些时候发现的Dbcn，人们对它知之甚少。目的1建议通过筛选额外的XLIS/双皮质突变来确定Dbcn蛋白的重要功能结构域。在目标2中，将确定Dbcn的mRNA和蛋白的分布，这是研究Dbcn可能的作用的必要的第一步。虽然数据库搜索发现很少有其他类似于Dbcn的蛋白质，但大脑中一个未知的蛋白质KIAA0369具有一个与Dbcn有约75%同源性的N-末端结构域和一个与CaM激酶有97%同源性的C-末端结构域。鉴于KIAA0369和Dbcn之间惊人的相似性以及它们具有相似(或竞争)功能的可能性，将确定KIAA0369相对于Dbcn的本地化。Dbcn、mDab和β-Lis1突变体的表型相似性表明，这些基因可能在相同的关键信号通路中发挥作用。这些蛋白质直接相互作用的可能性将在目标3中使用免疫沉淀和成对双杂交分析和免疫沉淀分析进行测试。非受体酪氨酸激酶，如Abl，可能参与皮层迁移的基本途径，和Walsh等人。已经在Dbcn中确定了一个可能的Abl磷酸化位点。Abl等非受体酪氨酸激酶失活蛋白的预测结构也支持了非受体酪氨酸激酶是这些通路的重要组成部分的假说。因此，目标3将包括确定Dbcn是否是Abl的底物，以及Abl是否与mDab1或beta Lis1相互作用的研究。这项建议的最后一个组成部分(目标4)是使用Dbcn、mDab1和beta Lis1进行一系列广泛的双杂交筛选，以筛选相互作用的蛋白质。任何被确认为相互作用的蛋白质都将在生化分析中进行结合测试，并将成为无脑畸形的候选基因。

DESCRIPTION: The proposal aims to identify and characterize components of the signaling pathways required for neuronal migration and cortical development. Although defects in Dbcn, mDab1, and beta LIS1 all lead to severe cortical abnormalities, little is known about the signaling pathways in which these gene products participate. Especially little is known about Dbcn, discovered earlier this year by Walsh and his colleagues. Aim 1 proposes to identify functionally important domains of the Dbcn protein by screening for additional XLIS/double cortex mutations. In aim 2 the distribution of Dbcn mRNA and protein will be determined, an essential first step to investigate the possible roles of Dbcn. Although database searches reveal few other proteins similar to Dbcn, an uncharacterized protein from brain known as KIAA0369 has an N-terminal domain that shares ~75% identity to Dbcn and a C-terminal domain that shares 97% identity with a cam kinase. Given the striking similarity between KIAA0369 and Dbcn and the possibility that they have similar (or competing) functions, the localization of KIAA0369 relative to Dbcn will be determined. The phenotypic similarity of mutants in Dbcn, mDab, and beta LIS1 suggests the hypothesis that these genes may function in the same critical signaling pathway. The possibility that these proteins interact directly with one another will be tested in aim 3 using immunoprecipitations and pair-wise two-hybrid assays and immunoprecipitation assays. Non-receptor tyrosine kinases such as Abl are likely to be involved in the pathways underlying cortical migration, and Walsh et al. have identified a putative Abl phosphorylation site in Dbcn. The predicted structure of Disabled as an adapter protein for non-receptor tyrosine kinases such as Abl also supports the hypothesis that non-receptor tyrosine kinases are important components of these pathways. Thus aim 3 will include studies to determine if Dbcn is a substrate for Abl and whether Abl interacts with mDab1 or beta LIS1. The final component of this proposal (aim 4) is to perform an extensive series of two-hybrid screens with Dbcn, mDab1, and beta LIS1 to screen for interacting proteins. Any proteins that are identified as interacting will be tested for binding in biochemical assays and would be candidate genes for lissencephaly.