NOVEL EFFECTORS OF GRB2 IN HUMAN GLIAL TUMORS

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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.