SIGNAL TRANSDUCTION PATHWAYS IN MEDULLOBLASTOMA

受体蛋白酪氨酸激酶（RPTKs）在 在人类髓母细胞瘤的发病机制中。 蛋白 含有SH 2和SH 3结构域的蛋白质对通过 RPTK。 我们最近克隆了Gab 1的cDNA（Grb 2相关 粘合剂-1）。 Gab 1作为一个对接蛋白的几个SH 2- 蛋白质包括PLC-γ、PI-3-激酶和SHPTP 2/syp， 是已知的促分裂信号。 Gab 1的过度表达 增强细胞生长并导致生长因子依赖性 转型 Gab 1在髓母细胞瘤中过度表达。 这个提议的中心假设是Gab 1有一个 在髓母细胞瘤发病机制中的重要作用 几个下游效应器的激活。 为了验证这一 假设，我们将执行以下操作： 在第一个具体目标中， 将测定Gab 1及其酪氨酸磷酸化程度 一系列的肿瘤。 Grb 2、PLC-γ、PI-3-β的比例 激酶，与Gab 2复合的SHPTP 2/syp将被 确定。 为了确定Gab 1的相对贡献， 还将评估EGF、IGF-1和Trk的这些参数 受体家族 细胞PI-3-激酶，PLC- 与Gab 1相关的γ和SHPTP 2/syp活性将 还可以被确定并与与以下相关联的活动进行比较： RPTK。 潜在的遗传改变或替代转录本 将评估Gab 1基因的突变。 在第二个具体目标中，我们将确定Gab 1是否是一个 IGF-1和TRK受体家族的底物，如果Gab 1 介导这些受体的酶活性。 重组Gab 1 将在体外激酶中用作这些RPTK的底物 测定。 Gab 1的磷酸化反应 将对增长进行评估。 还将确定， 这些受体的磷酸化介导了 PLC-γ、PI-3-激酶和SHPTP 2/syp对Gab 1的活性。 任何 这些分子在招募Gab 1时的差异， 将确定这些受体诱导的。 在第三个具体目标中，我们将通过 使用显性负突变体。 Gab 1上的磷酸酪氨酸 作为PI-3的SH 2结构域的识别位点， 激酶、PLC-γ和SHPTP 2/syp将被定位。 肽 将进行竞争，以确认这些的真实性 网站. 这些位点将被突变，看看这是否会废除Gab 1 体内相关酶活性。 的后果 这些突变体在转化的表型上的过表达 将确定成神经管细胞瘤细胞系。 这将产生 了解肿瘤细胞需要哪些特定分子 增长

Receptor protein tyrosine kinases (RPTKs) have an essential role in the pathogenesis of human medulloblastoma tumors. Proteins containing SH2 and SH3 domain are crucial to the signaling by RPTKs. We recently cloned the cDNA for Gab1 (Grb2 associated binder-1). Gab1 acts as a docking protein for several SH2- proteins including PLC-gamma, PI-3-kinase, and SHPTP2/syp, which are known to transduce mitogenic signals. Overexpression of Gab1 enhances cell growth and results in growth factor dependent transformation. Gab1 is overexpressed in medulloblastoma tumors. The central hypothesis of this proposal is that Gab1 has an important role in medulloblastoma tumor pathogenesis by enhancing the activation of several downstream effectors. To test this hypothesis, we will perform the following: In the first specific aim, the relative levels of expression of Gab1 and its degree of tyrosine phosphorylation will be determined in series of tumors. The proportion of Grb2, PLC-gamma, PI-3- kinase, and SHPTP2/syp that is complexed to Gab2 will be ascertained. To determine the relative contribution of Gab1, these parameters will also be assessed for the EGF, IGF-1 and Trk family of receptors. The amount of cellular PI-3-kinase, PLC- gamma and SHPTP2/syp activity that is associated with Gab1 will also be determined and compared to the activity associated with RPTKs. Potential genetic alterations or alternative transcripts from the Gab1 gene will be evaluated. In the second specific aim, we will determine if Gab1 is a substrate of the IGF-1 and TRK family of receptors and if Gab1 mediates enzymatic activity for these receptors. Recombinant Gab1 will be used as a substrate for these RPTKs in in vitro kinase assays. The phosphorylation of Gab1 in response to the cognate growth will be evaluated. It will also be determined if the phosphorylation by these receptors mediates the recruitment of PLC-gamma, PI-3-kinase and SHPTP2/syp activity to Gab1. Any differences in the recruitment of these molecules to Gab1 as induced by these receptors will be determined. In the third specific aim, we will probe Gab1 function through the use of dominant negative mutants. The phosphotyrosine on Gab1 that serve as recognition sites for the SH2 domains of PI-3- kinase, PLC-gamma and SHPTP2/syp will be mapped. Peptide competition will be performed to confirm the authenticity of these sites. These sites will be mutated to see if this abolishes Gab1 associated enzymatic activity in vivo. The consequences of the overexpression of these mutants on the transformed phenotype of medulloblastoma cell lines will be determined. This will yield insights into which specific molecules are required for tumor cell growth.