Membrane targeting of fatty acylated proto-oncoproteins

描述（由申请人提供）：本研究的总体目标是了解蛋白质脂肪酰化和膜胆固醇在指导信号转导蛋白亚细胞定位和功能中的作用。酪氨酸蛋白激酶Src家族的成员将被用作模型系统。具体地，本申请中概述的实验被设计为解决以下问题： 1. 确定细胞膜胆固醇耗竭激活的信号分子和途径。我们最近发现，用环糊精耗竭膜胆固醇可激活MAP激酶，并导致许多蛋白质酪氨酸磷酸化增加。将确定这些蛋白质的身份，以及MAPK活化和蛋白质酪氨酸磷酸化之间的关系。将使用药理学和显性负性蛋白抑制剂鉴定被激活的细胞内信号传导途径。 2. 确定Src家族激酶（SFK）脱棕榈酰化的动力学和调节。棕榈酰化是控制蛋白质定位和信号传导的动态反应。我们将确定是否APT 1，一种新描述的硫酯酶，depalmitoylates SFKs和SFK depalmitoylation是否由筏定位和/或信号转导调节。 3. 确定Src家族激酶利用的细胞内运输机制。将使用GFP标记的SFKs的共聚焦显微镜和延时成像的组合来确定参与将SFKs引导至特定膜位置的亚细胞运输途径。我们将测试SFKs是否与内吞机制的组分相互作用，从而到达质膜。

DESCRIPTION (provide by applicant): The overall goal of this research is to understand the role of protein fatty acylation and membrane cholesterol in directing subcellular localization and function of signal transduction proteins. Members of the Src family of tyrosine protein kinases will be used as model systems. Specifically, the experiments outlined in this application are designed to address the following: 1. To identify signaling molecules and pathways activated by plasma membrane cholesterol depletion. We recently showed that depletion of membrane cholesterol with cyclodextrin activates MAP kinase and causes increased tyrosine phosphorylation on a number of proteins. The identity of these proteins will be determined, as well as the relationship(s) between MAPK activation and protein tyrosine phosphorylation. The intracellular signaling pathways that are activated will be identified using pharmacological and dominant negative protein inhibitors. 2. To determine the kinetics and regulation of Src family kinase (SFK) depalmitoylation. Palmitoylation is a dynamic reaction that controls protein localization and signaling. We will determine whether APT1, a newly described thioesterase, depalmitoylates SFKs and whether SFK depalmitoylation is regulated by raft localization and/or signal transduction. 3. To identify the intracellular trafficking mechanism(s) utilized by Src family kinases. The subcellular trafficking pathway(s) involved in directing SFKs to specific membrane locations will be determined using combinations of confocal microscopy and time-lapse imaging of GFP-tagged SFKs. We will test whether SFKs interact with components of the endocytic machinery en route to the plasma membrane.