Biochemical screen--regulators of neuronal morphogenesis

描述(由申请人提供)：蛋白酪氨酸激酶是轴突和树突引导线索的重要中介。在大多数情况下，调节这些激酶下游作用的蛋白质靶标仍有待确定。我们研究的目标是采用一种生化技术，即“凹坑”方法，来识别发育中的大脑中的酪氨酸激酶底物。Abl和Arg非受体酪氨酸激酶是发育中的小鼠大脑皮质树突分支形成所必需的。作为概念的证明，我们建议在发育中的大脑中识别和功能表征Abl和Arg的底物。我们的第一个目标是在发育中的神经元中鉴定Abl和Arg底物。我们已经合成了一种经过化学改变的ATP，它在ATP的腺苷环上含有一个苄基“凹凸”，并通过从ATP结合口袋中移除一个笨重的氨基酸残基，在Abl和Arg上设计了一个相应的“洞”。重要的是，这些改变的特异性(AS-)形式的Abl和Arg可以利用碰撞的ATP，而存在于脑提取液中的激酶则不能。我们将在体外选择性地标记Abl和Arg底物，方法是将脑提取物与As-Abl或As-Arg在Gamma32P标记的凹凸不平的ATP存在下孵育。我们将使用生化分离和抗磷酸酪氨酸亲和树脂来纯化标记底物，并用质谱仪对它们进行鉴定。我们将使用体外和基于细胞的磷酸化试验来确认所确定的候选是真正的Abl/Arg底物。我们的第二个目标是确定底物是否调节轴突或树突的形态发生。我们将研究每种底物在培养的野生型和Arg缺陷型皮质神经元中的定位，并确定整合素介导的黏附或Arg激酶活性升高如何影响这种定位。我们将测试底物的过度表达是否单独或与精氨酸联合影响培养的皮质神经元的轴突或树突分支。我们将确定RNAi介导的调节因子的减少如何影响黏附或Arg过表达后的轴突和树突分支。这些研究应该证明使用凹坑方法在发育中的神经元中识别酪氨酸激酶靶标的实用性。识别这些底物将扩展可用于剖析轴突和树突形态发生的分子机制的“工具箱”。

DESCRIPTION (provided by applicant): Protein tyrosine kinases are essential mediators of axon and dendrite guidance cues. In most cases, the protein targets that mediate the downstream actions of these kinases remain to be identified. The goal of our research is to adapt a biochemical technique, the "bump-hole" approach, to identify tyrosine kinase substrates in the developing brain. The Abl and Arg nonreceptor tyrosine kinase are required for cortical dendrite branch formation in the developing mouse brain. As a proof of concept, we propose to identify and functionally characterize substrates of Abl and Arg in the developing brain. Our first aim is to identify Abl and Arg substrates in developing neurons. We have synthesized a chemically altered form of ATP that contains a benzyl group "bump" on the adenosine ring of ATP (bumped ATP) and engineered a corresponding "hole" in Abl and Arg by removing a bulky amino acid residue from the ATP-binding pocket. Importantly, these altered specificity- (as-) forms of Abl and Arg can utilize the bumped ATP, whereas kinases present in brain extracts cannot. We will selectively label Abl and Arg substrates in vitro by incubating brain extracts with as-Abl or as-Arg in the presence of gamma32P-labeled bumped ATP. We will purify labeled substrates using biochemical fractionation and anti-phosphotyrosine affinity resins and identify them by mass spectrometry. We will use in vitro and cell-based phosphorylation assays to confirm that the identified candidates are bona fide Abl/Arg substrates. Our second aim is to determine whether the substrates regulate axon or dendrite morphogenesis. We will examine the localization of each substrate in cultured wild type and Arg-deficient cortical neurons and determine how this localization is influenced by integrin-mediated adhesion or elevated Arg kinase activity. We will test whether overexpression of the substrate alone or in combination with Arg affects axon or dendrite branching in cultured cortical neurons. We will determine how RNAi-mediated reduction of the regulator influences axon and dendrite branching following adhesion or Arg overexpression. These studies should prove the utility of using the bump-hole approach to identify tyrosine kinase targets in developing neurons. Identifying these substrate will expand the "toolkit" available to dissect the molecular mechanisms of axon and dendrite morphogenesis.