Membrane targeting of fatty acylated proto-oncoproteins

脂肪酰化原癌蛋白的膜靶向

• 批准号: 6545388
• 负责人: MARILYN D RESH
• 金额: $ 26.78万
• 依托单位: SLOAN-KETTERING INST CAN RESEARCH
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-08-01 至 2006-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6545388
• 关键词: 3T3 cells; SDS polyacrylamide gel electrophoresis; biological signal transduction; cell membrane; cholesterol; confocal scanning microscopy; fatty acylation; green fluorescent proteins; image processing; membrane fusion; membrane lipids; mitogen activated protein kinase; oncoproteins; phosphorylation; protein localization; protein structure function; protein transport; protein tyrosine kinase; protooncogene; western blottings

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.

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的亚细胞定位和功能中的作用。酪氨酸蛋白激酶 Src 家族的成员将用作模型系统。具体来说，本申请中概述的实验旨在解决以下问题： 1. 鉴定质膜胆固醇消耗所激活的信号分子和途径。我们最近表明，用环糊精消耗膜胆固醇会激活 MAP 激酶，并导致许多蛋白质的酪氨酸磷酸化增加。将确定这些蛋白质的身份，以及 MAPK 激活和蛋白质酪氨酸磷酸化之间的关系。将使用药理学和显性失活蛋白抑制剂来鉴定被激活的细胞内信号传导途径。 2. 确定Src家族激酶(SFK)去棕榈酰化的动力学和调节。棕榈酰化是一种控制蛋白质定位和信号传导的动态反应。我们将确定 APT1（一种新描述的硫酯酶）是否能够使 SFK 去棕榈酰化，以及 SFK 去棕榈酰化是否受到筏定位和/或信号转导的调节。 3. 鉴定Src家族激酶利用的细胞内运输机制。将使用共聚焦显微镜和 GFP 标记的 SFK 的延时成像相结合来确定将 SFK 引导至特定膜位置所涉及的亚细胞运输途径。我们将测试 SFK 是否在通往质膜的途中与内吞机制的组件相互作用。