A biochemical screen for novel 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导向信号的基本介质。在大多数情况下，介导这些激酶下游作用的蛋白质靶点仍有待鉴定。我们的研究目标是采用一种生物化学技术，即“碰撞孔”方法，来识别发育中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需的。作为一个概念的证明，我们提出了识别和功能特性的Abl和Arg在发育中的大脑基板。我们的第一个目标是确定Abl和Arg底物在发育中的神经元。我们已经合成了一种化学改变形式的ATP，其在ATP的腺苷环上含有苄基“凸点”（凸点ATP），并通过从ATP结合口袋中去除庞大的氨基酸残基来在Abl和Arg中设计相应的“孔”。重要的是，Abl和Arg的这些改变的特异性形式可以利用撞击的ATP，而脑提取物中存在的激酶不能。我们将选择性地标记Abl和Arg底物在体外孵育脑提取物与as-Abl或as-Arg在存在的γ 32 P-标记的凹凸ATP。我们将纯化标记的底物使用生化分馏和抗磷酸酪氨酸亲和树脂，并确定它们的质谱。我们将使用体外和基于细胞的磷酸化测定来确认所鉴定的候选物是真正的Abl/Arg底物。我们的第二个目的是确定是否基板调节轴突或树突形态发生。我们将研究在培养的野生型和精氨酸缺陷的皮质神经元中的每种底物的定位，并确定这种定位是如何受到整合素介导的粘附或升高的精氨酸激酶活性的影响。我们将测试是否过度表达的基板单独或与精氨酸的组合影响轴突或树突在培养的皮层神经元的分支。我们将确定RNAi介导的调节剂的减少如何影响粘附或Arg过表达后的轴突和树突分支。这些研究应该证明使用凹凸孔方法来识别发育中的神经元中的酪氨酸激酶靶点的实用性。确定这些基板将扩大“工具箱”可用于解剖轴突和树突形态发生的分子机制。

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.