Oncogene-mediated transformation results in the disruption of normal cellular growth-control. Understanding the role normal counterparts of transforming agents play in cellular homeostasis will enhance our ability to decipher the molecular mechanisms by which oncogenic agents cause this system to go awry. Our studies have provided two lines of evidence which suggest that the proto- oncogene product, pp60c-src, is involved in the mitogenic response of cells to epidermal growth factor (EGF). First, murine C3H10T1/2 fibroblasts constitutively overexpressing pp60c-src exhibit a hyper-responsiveness to EGF (as measured by 3H- thymidine incorporation) when compared to the response of stimulated, parent cells. Second, a dramatic negative regulation of pp60c-src specific kinase activity occurs in quail embryo fibroblasts transformed by the avian erythroblastosis virus (AEV), which encodes aberrant forms of the thyroxin receptor (v-erb A) and the EGF receptor (v-erb B). The experiments outlines in this proposal have two inter-related aims. The first is to determine the molecular mechanism for the enhanced responsiveness of c- src overexpressors to EGF by analyzing and comparing levels of second messengers, critical protein kinases and expression of proto-oncogenes in mitogen-stimulated parent and overexpressor lines. Analysis of cells overexpressing c-src bearing site-directed mutations in the molecule and microinjection of monoclonal antibodies with specificities to defined epitopes will be carried out to determine regions within the c-src protein which are required for the interaction with cellular components that result in the enhanced response to growth factors.
The second aim i s to determine the mechanism of down-modulation of pp60c-src activity in AEV infected cells. A genetic approach utilizing deletion and temperature-sensitive mutants of AEV will allow us to determine whether v-erb A or v-erb B or both are required for this effect. Intracellular localization studies, analysis of signal transduction components, and purification and identification of cellular agents which mediate the regulatory effects on pp60c-src in uninfected and wild type and mutant infected AEV cells are also proposed. Together these studies have as their common goal the elucidation of the temporal and spatial relationships of pp60c- src to substrate and regulatory components of the EGF-dependent signal transduction pathway(s).
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