While tyrosine kinases are clearly established as primary signaling molecules for a variety of growth factor receptors and oncogene products, the identity and mechanism of their phosphorylation substrates is only beginning to be understood. Phospholipase C-gamma1 (PLC-gamma1) is a substrate for several tyrosine kinases including the epidermal growth factor (EGF) receptor. This application proposes three types of studies (biochemical, biophysical, and biological) to understand the interaction of PLC-gamma1 with EGF receptor. Biochemical studies constitute the major thrust of this project. These studies are proposed to understand the mechanism by which tyrosine phosphorylation of PLC-gamma1 by the EGF receptor increases the catalytic activity of this enzyme. Variables such as phospholipid composition of substrate vesicles and the presence of counterions will be tested for the tyrosine phosphorylated form of PLC-gamma1 and the non-phosphorylated forms of PLC-gamma1. Also, the influence of other proteins (PIP2 binding proteins, membrane components, and tyrosine phosphatases) will be compared. Mechanistic issues will also be explored. The intent of these studies is to develop a complete in vitro system for understanding the nature of activation of PLC-gamma1 by tyrosine phosphorylation. Biophysical approaches are planned to measure phosphorylation-dependent changes in PLC-gamma1 conformation and to provide rapid and detailed measurements for different interactions between PLC-gamma1 and other proteins or lipid substrates. Biological studies will attempt to determine whether PLC-gamma1 activation is effected by EGF in cell systems where EGF produces a non-mitogenic response. Additional biological studies will utilize mitogenic response cell line to analyze PLC-gamma1 phosphorylation during the cell cycle and during the transition from Gomicron to S.
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