NOVEL EFFECTORS OF GRB2 IN HUMAN GLIAL TUMORS

我们已经确定了一种新的参与信号转导的分子，称为GAB1，并表明它作为EGF和胰岛素受体的对接蛋白发挥作用。我们的实验室和其他实验室的工作现在已经证明，GAB1在信号转导中发挥了更大的作用。至少有6个受体蛋白酪氨酸激酶(RPTKs)和4个细胞因子受体在配体激活后磷酸化GAB1。GAB1结合多个SH2蛋白，包括PI3K和SHP-2，以及PI3K的脂类产物PIP3。这些相互作用促进了MAPK的激活、细胞的存活和转化。我们最近发现，GAB1也是连接G蛋白偶联受体(GPCRs)和通过PI3-激酶激活MAP激酶的关键成分。在这个项目期间，我们建议进一步定义GAB1在信号转导中的作用，并表征最近发现的一个相关的对接蛋白，称为GABF。为了继续我们对GPCR信号的研究，我们将确定GAB1与SHP-2的相互作用如何有助于MAP激酶的激活。我们将利用GAB1的显性负性突变体来分析GAB1的PH结构域在结合PIP3和G蛋白的Betagamma亚基中的作用。由于GAB1激活PI3-激酶，我们还将评估GAB1在GPCR诱导的细胞骨架重排中的作用。同样，将确定GAB1是否增强了由GPCR刺激启动的细胞运动。要研究GAB1在生物体中的作用，最好的方法是通过敲除小鼠。我们已经产生了一个靶向结构，并将继续产生纯合子基因破坏的小鼠。然后将对这些小鼠进行任何发育和/或病理异常的研究。成纤维细胞将从GAB1-/-和GAB1+/-小鼠中分离出来，用于检测GAB1在RPTK介导的转化中的作用以及GAB1在信号转导途径中的作用。我们最近克隆了GABF，发现它与GAB1高度相关。虽然这两种蛋白在EGF刺激下都被磷酸化，与SHP-2结合，并与SH2-蛋白有许多其他结合部位，但还有一些其他的区别。我们将通过鉴定GPCR、细胞因子和其他使GABF磷酸化的RPTK受体来进一步表征GABF在信号转导中的作用。将确定GABF是否增加了与转化相关的表型。与GABF结合的Sh2-蛋白将被鉴定，GABF上的结合位点将发生突变。然后将评估表达这些突变的cDNA对转化的影响。由于GABF不能与PIP3结合，我们将研究这是否会影响膜的定位。最后，我们将通过评估GABF的磷酸化状态和肿瘤裂解物中SH2蛋白的相关性，以及GABF在肿瘤切片中的免疫组织化学定位，来评估GABF在原发胶质肿瘤中的潜在作用。

We have identified a new molecule involved in signal transduction called Gab1 and have shown that it functions as a docking protein for the EGF and insulin receptors. Work by our lab and others have now demonstrated a much greater role for Gab1 in signaling. At least 6 other receptor protein tyrosine kinases (RPTKs) and 4 cytokine receptors phosphorylate Gab1 after ligand activation. Gab1 binds several SH2-proteins, including PI 3-kinase and SHP-2, and the lipid product of PI 3-kinase, PIP3. These interactions promote MAP kinase activation, cell survival and transformation. We have recently found that Gab1 is also a critical component in linking G-protein-coupled receptors (GPCRs) to the activation of MAP kinase via activation of PI 3-kinase. In this project period, we propose to further define the role of Gab1 in signaling and to characterize a recently discovered related docking protein called GabF. To continue our studies in GPCR signaling we will determine how the Gab1 interaction with SHP-2 contributes to MAP kinase activation. The role of the PH domain of Gab1 in binding PIP3 and the betagamma subunits of G-proteins will be analyzed through the use of dominant negative mutants of Gab1. Since Gab1 activates PI 3-kinase, we will also evaluate the role of Gab1 in GPCR-induced rearrangements of the cytoskeleton. Similarly, it will be determined if Gab1 enhances cell motility initiated by GPCR stimulation. The best method to study the role of Gab1 in the context of an organism is via a knock out mouse. We have already generated a targeting construct and will proceed to generate mice with a homozygous disruption of the gene. These mice will then be studied for any developmental and/or pathologic abnormalities. Fibroblasts will be derived from Gab1 -/- and Gab1 +/- mice and used to test the role of Gab1 in RPTK-mediated transformation and the contribution of Gab1 to signal transduction pathways. We recently cloned GabF and found that it is highly related to Gab1. While both proteins are phosphorylated upon EGF stimulation, bind SHP-2 and share many other binding sites for SH2-proteins, there are some other differences. We will further characterize the role of GabF in signal transduction by identifying GPCR, cytokine and other RPTK receptors that phosphorylate GabF. It will be determined if GabF augments phenotypes related to transformation. SH2-proteins that bind to GabF will be identified and the binding sites on GabF will be mutated. The effects of expressing these mutated cDNAs on transformation will then be assessed. Since GabF fails to bind PIP3 we will study if this affects membrane localization. Finally, we will evaluate the potential role of GabF in primary glial tumors by assessing GabF phosphorylation status and the association of SH2-proteins in tumor lysates, and GabF localization in tumor sections by immunohistochemistry.