Novel Effectors of GRB2 in Human Glial Tumors

描述（由申请人提供）：Gab 1是一种对接蛋白，位于多种受体酪氨酸激酶（RTK）的下游。本研究的目的是进一步了解Gab 1在EGF受体和活性氧（reactive oxygen species，ROS）信号转导中的作用，以及Gab 1如何参与胶质瘤的发生。我们已经表明，Gab 1可以是SHP-2酪氨酸磷酸酶的底物，这种相互作用是必不可少的细胞生长和转化NIH 3 T3细胞。相反，在氧化应激下，我们发现Gab 1/SHP-2复合物促进细胞死亡，并与JNK激活相关。我们第一次观察到Gab 1在高级别星形细胞瘤肿瘤中过表达，与正常胶质细胞相比，提示Gab 1在胶质瘤发生中的作用。它还呈现两种不同的定位模式，一种是弥漫性的，另一种是在外周的不寻常的囊泡染色，其频率随着肿瘤分级而增加。在具体目标#1中，我们将通过以下方式研究Gab 1在细胞生长和转化中的作用：1）分析SHP-2脱磷酸化的Gab 1位点; 2）使用此信息识别负责细胞生长和/或转化的Gab 1/SHP 2相互作用下游的途径。具体目标#2将通过以下方式关注其在ROS刺激中的作用：1）通过FRET和细胞分级研究氧化应激后Gab 1/SHP-2相互作用的定位; 2）MS-MALDI显示的H2 O2添加后Gab 1的磷酸化位点; 3）JNK如何通过Gab 1/SHP-2相互作用特异性激活。在具体目标#3中，我们将通过研究以下内容来检查Gab 1在星形细胞瘤中的作用：1）在III级和IV级细胞系中Gab 1表达的敲低是否会损害致瘤性; 2）Gab 1的存在是否与次级途径引起的IV级肿瘤相关;以及3）Gab 1所在的囊泡样结构的性质。这项工作将应用尚未在癌细胞中广泛使用的最先进的工具，并可能揭示细胞定位如何与肿瘤中信号传导分子的信号传导和独特性质相关的新方面。

DESCRIPTION (provided by applicant): Gab1 is a docking protein that is downstream of multiple receptor tyrosine kinases (RTKs). Our goals are to further understand the role of Gab1 in EGF receptor and reactive oxygen species (ROS) signaling and how it participates in the process of gliomagenesis. We have shown that Gab1 can be a substrate for the SHP-2 tyrosine phosphatase and this interaction is essential for cell growth and transformation in NIH3T3 cells. In contrast, under oxidative stress we find that the Gab1/SHP-2 complex promotes cell death and is correlated with JNK activation. For the first time, we have observed that Gab1 is overexpressed in high grade astrocytoma tumors in comparison with normal glia, suggesting a role for Gab1 in gliomagenesis. It also presents two different patterns of localization, one diffuse and the other an unusual vesicular staining at the periphery which increases in frequency with tumor grade. In Specific Aim #1, we will study the role of Gab1 in cell growth and transformation by: 1) analyzing the sites in Gab1 dephosphorylated by SHP-2; 2) using this information to identify the pathways downstream of the Gab1/SHP2 interaction responsible for cell growth and/or transformation. Specific Aim #2 will focus on its role in ROS stimulation by :1) studying the localization of Gab1/SHP2 interaction after oxidative stress via FRET and cell fractionation; 2) the phosphorylation sites of Gab1 after H2O2 addition as revealed by MS-MALDI; and 3) how JNK is specifically activated by the Gab1/SHP-2 interaction. In Specific Aim #3, we will examine the role of Gab1 in astrocytomas by studying: 1) if knock down of Gab1 expression in grade III and grade IV cell lines impairs tumorigenicity; 2) if the presence of Gab1 correlates with grade IV tumors arising from the secondary pathway; and 3) what is the nature of the vesicle-like structure where Gab1 is localized. This work will apply state of the art tools that have not been extensively used in cancer cells and may reveal new aspects of how cellular localization correlates with signaling and unique properties of signaling molecules in tumors.