NOVEL EFFECTORS OF GRB2 IN HUMAN GLIAL TUMORS

我们已经确定了一个新的分子参与信号转导称为Gab 1，并已表明，它的功能作为对接蛋白的EGF和胰岛素受体。 我们实验室和其他人的工作现在已经证明了Gab 1在信号传导中的重要作用。配体激活后，至少有6种其他受体蛋白酪氨酸激酶（RPTK）和4种细胞因子受体磷酸化Gab 1。Gab 1结合几种SH 2蛋白，包括PI 3-激酶和SHP-2，以及PI 3-激酶的脂质产物PIP 3。 这些相互作用促进MAP激酶活化、细胞存活和转化。我们最近发现，Gab 1也是连接G蛋白偶联受体（GPCRs）通过PI 3-激酶的激活MAP激酶的激活的关键组成部分。 在这个项目期间，我们建议进一步确定Gab 1在信号转导中的作用，并描述最近发现的一种相关的对接蛋白GabF。为了继续我们在GPCR信号转导中的研究，我们将确定Gab 1与SHP-2的相互作用如何有助于MAP激酶的激活。 Gab 1的PH结构域在结合PIP 3和G蛋白的β γ亚基中的作用将通过使用Gab 1的显性负突变体来分析。 由于Gab 1激活PI 3-激酶，我们还将评估Gab 1在GPCR诱导的细胞骨架重排中的作用。 类似地，将确定Gabl是否增强由GPCR刺激引发的细胞运动性。研究Gab 1在生物体中作用的最佳方法是通过敲除小鼠。 我们已经产生了一个靶向构建体，并将继续产生基因纯合破坏的小鼠。 然后研究这些小鼠的任何发育和/或病理异常。成纤维细胞将来自Gab 1-/-和Gab 1 +/-小鼠，并用于测试Gab 1在RPTK介导的转化中的作用以及Gab 1对信号转导途径的贡献。我们最近克隆了GabF，发现它与Gab 1高度相关。 虽然这两种蛋白质在EGF刺激下磷酸化，结合SHP-2并共享许多其他SH 2蛋白结合位点，但存在一些其他差异。 我们将通过鉴定磷酸化GabF的GPCR、细胞因子和其他RPTK受体来进一步表征GabF在信号转导中的作用。 将确定GabF是否增加与转化相关的表型。 将鉴定与GabF结合的SH 2蛋白，并突变GabF上的结合位点。 然后将评估表达这些突变的cDNA对转化的影响。 由于GabF不能结合PIP 3，我们将研究这是否影响膜定位。最后，我们将通过评估GabF磷酸化状态和肿瘤裂解物中SH 2蛋白的相关性，以及通过免疫组织化学在肿瘤切片中的GabF定位，来评估GabF在原发性神经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.