The long term goals of the experiments proposed in this application are to understand the functional consequences of tyrosine phosphorylation of tyrosine protein kinase substrates and the role of these substrates in the molecular signalling pathways induced by non-receptor tyrosine kinases and by ligand activated receptor tyrosine kinases. The proposed experiments build on the progress made during the past granting period in identifying novel substrates for pp60src and the epidermal growth factor (EGF) receptor. We will focus on the functional analysis of three tyrosine kinase substrates that appear to play important and perhaps novel roles in the mitogenic signalling process. In addition we will investigate the role of the ras gene product, p21ras, in the cascade of events mediated by receptor tyrosine protein kinases.
The specific aims are: 1) To functionally characterize an 80 kDa tyrosine kinase substrate (TKS/80), recently identified in our laboratory as an actin binding protein. This analysis will include identification of the sites of phosphorylation within TKS/80, a study of its association with other cellular proteins, and an investigation f its role in pp60src and EGF receptor mediated signalling. 2) Purification and analysis of SH2bp130, a pp60src SH2 domain binding protein. These experiments will include the isolation of additional antibodies to SH2bp130, isolation of cDNAs encoding SH2bp130, and a functional characterization of SH2bp130 in cell transformation and EGF mediated signalling. 3) Purification and analysis of GAPbp64, a 64 kDa protein that associates with GTPase activating protein (GAP) in src transformed and EGF stimulated cells. This analysis will include the isolation of antibodies to GAPbp64, isolation of GAPbp64 cDNAs and a functional characterization of its interactions with EGF receptor, pp60src, and GAP.4) The dominant inhibitory mutant of H-ras, Asn17 will be used to assess the role of p21ras in receptor mediated tyrosine phosphorylation of cellular substrates. These experiments will test the hypothesis that functional p21ras is required for the efficient mobilization of components within certain signal transduction pathways. Together these studies will shed light on the relationship between receptor activation, tyrosine phosphorylation, activation of signalling pathways and the regulation of cell growth.
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