SIGNAL TRANSDUCTION PATHWAYS IN MEDULLOBLASTOMA

受体蛋白酪氨酸激酶(RPTKs)具有重要的作用 在人类髓母细胞瘤的发病机制中起重要作用。蛋白质 含有SH2和SH3结构域对信号转导起关键作用 RPTKs。我们最近克隆了GAB1(Grb2相关的 粘合剂-1)。GAB1是几种SH2-的对接蛋白 蛋白质包括PLC-γ、PI-3-激酶和SHPTP2/SYP，它们 都是已知的转导有丝分裂信号。GAB1基因的过表达 促进细胞生长并导致生长因子依赖 转型。GAB1在髓母细胞瘤中过表达。 这一提议的中心假设是GAB1有一个 增强基因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。为了测试这一点 假设，我们将执行以下操作： 在第一个具体目标中，表达的相对水平 将测定GAB1及其酪氨酸磷酸化程度 在一系列肿瘤中。Grb2、PLC-伽马、PI-3的比例 与Gab2络合的SHPTP2/SYP将是 已经确定了。为了确定Gab1的相对贡献， 这些参数也将针对EGF、IGF-1和Trk进行评估 受体家族。细胞内PI-3-激酶、PLC- 与GAB1关联的Gamma和SHPTP2/SYP活动将 也可以被确定，并与与 RPTKs。潜在的基因改变或替代转录本 将对来自GAB1基因的基因进行评估。 在第二个具体目标中，我们将确定GAB1是否是 IGF-1和TRK受体家族的底物和IF GAB1 介导这些受体的酶活性。重组GAB1 将被用来作为这些RPTKs的底物在体外激酶 化验。GAB1对同源基因的磷酸化反应 将对增长进行评估。还将确定是否 这些受体的磷酸化介导了细胞的募集 PLC-γ、PI-3-激酶和SHPTP2/SYP活性对GAB1的影响。任何 这些分子在GAB1上的招募差异 这些受体的诱导作用将会被确定。 在第三个具体目标中，我们将通过 显性负变异体的使用。GAB1上的磷酸酪氨酸 作为PI-3-SH2结构域的识别位点 激酶、PLC-伽马和SHPTP2/SYP将被映射。多肽 将进行竞赛以确认这些材料的真实性 网站。这些位点将发生突变，看看这是否会废除GAB1 体内相关的酶活性。这场战争的后果 这些突变体在转化的表型上的过表达 将确定髓母细胞瘤细胞系。这将会产生 对肿瘤细胞需要哪些特定分子的洞察 成长。

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.