Gab1 is a docking protein that is downstream of multiple receptor tyrosine kinases (RTKs). Our goals are to further understand the role of Gab1 in EGF receptor and reactive oxygen species (ROS) signaling and how it participates in the process of gliomagenesis. We have shown that Gab1 can be a substrate for the SHP-2 tyrosine phosphatase and this interaction is essential for cell growth and transformation in NIH3T3 cells. In contrast, under oxidative stress we find that the Gab1/SHP-2 complex promotes cell death and is correlated with JNK activation. For the first time, we have observed that Gab1 is overexpressed in high grade astrocytoma tumors in comparison with normal glia, suggesting a role for Gab1 in gliomagenesis. It also presents two different patterns of localization, one diffuse and the other an unusual vesicular staining at the periphery which increases in frequency with tumor grade.
In Specific Aim #1, we will study the role of Gab1 in cell growth and transformation by: 1) analyzing the sites in Gab1 dephosphorylated by SHP-2; 2) using this information to identify the pathways downstream of the Gab1/SHP2 interaction responsible for cell growth and/or transformation.
Specific Aim #2 will focus on its role in ROS stimulation by :1) studying the localization of Gab1/SHP2 interaction after oxidative stress via FRET and cell fractionation; 2) the phosphorylation sites of Gab1 after H2O2 addition as revealed by MS-MALDI; and 3) how JNK is specifically activated by the Gab1/SHP-2 interaction.
In Specific Aim #3, we will examine the role of Gab1 in astrocytomas by studying: 1) if knock down of Gab1 expression in grade III and grade IV cell lines impairs tumorigenicity; 2) if the presence of Gab1 correlates with grade IV tumors arising from the secondary pathway; and 3) what is the nature of the vesicle-like structure where Gab1 is localized. This work will apply state of the art tools that have not been extensively used in cancer cells and may reveal new aspects of how cellular localization correlates with signaling and unique properties of signaling molecules in tumors.
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