Receptor protein tyrosine kinases (RPTKs) are essential to the pathogenesis of human glial tumors. The applicant recently cloned the cDNA for Gab1 (Grb2 associated binder-1) which links RPTKs to SH2/SH3 domain containing proteins. Gab1 is a docking protein for SH2-proteins including Grb2, PLC-gamma, PI-3-kinase and SHPTP2/syp, proteins known to transduce mitogenic signals. Overexpression of Gab1 in NIH-3T3 cells results in transformation. Gab1 is overexpressed in primary glial tumors. It is hypothesized that Gab1 has an important role in glial tumor pathogenesis by enhancing the activation of several mitogenic downstream effectors. It will be determined which SH2-proteins are essential for the transforming effects of Gab1. Phosphotyrosines on Gab1 that serve as recognition sites for the SH2 domains of Grb2, PI-3-kinase, PLC-gamma and SHPTP2/syp will be ascertained. Peptide competition will be performed to confirm this and the sites will be mutated to see if this abolishes binding to Gab1 and associated enzymatic activity. The consequences of the oeverexpression of these mutants on various aspects of the transformed phenotype in NIH-3T3 cells will be determined. The role of the PH domain and the proline rich binding sites for Grb2 will be assessed by deleting these regions and assaying for any changes in SH2-protein activity as well as transformation related parameters. Next, it will be addressed if Gab1 is directly downstream of three RPTKs known to be expressed in glial tumors: the PDGF, IGF-1 and TrkA receptors. Recombinant Gab1 will be used as a substrate for these receptors in an in vitro kinase assay. This will be confirmed in intact cells by treating with the cognate growth factors and looking for tyrosine phosphorylation of Gab1. It will also be determined if phosphorylation by these receptors mediates the recruitment of PLC-gamma, PI-3-kinase and SHPTP2/syp activity to Gab1. Finally, the role of GAB1 in primary human glial tumors will be examined. The relative levels of expression of GAB1 and its degree of tyrosine phosphorylation will be assayed in a series of primary glial tumors. The proportion of endogenous GRB2, PLC-gamma, PI-3-kinase and SHPTP2/syp that is complexed to Gab1 will be ascertained as well as the amount associating with RPTKs and anti-phosphotyrosine antibody. The actual enzymatic activity associated with Gab1 will be quantitated and compared to that associated with RPTKs and anti-phosphotyrosine antibody. The structure of alternative transcripts from the Gab1 gene in tumors will be determined and their effects on transformation and signaling will be evaluated.
