SIGNAL TRANSDUCTION PATHWAYS IN MEDULLOBLASTOMA

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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.