An increasing body of evidence suggests that the products of normal cellular oncogenes (proto-oncogenes) participate in the biochemical mechanisms by which cells regulate their own growth and differentiation. The mechanism(s) by which viral oncogene products or structurally altered cellular oncogene products disrupt the activity and functional interactions of normal cellular proteins involved in growth regulation remains unclear. Our preliminary evidence suggests that the activity of the product of the src proto-oncogene, pp60c-src, is modulated in cells transformed by a variety of agents, including retroviruses, DNA tumor viruses, and chemical mutagens. In addition, a similar modulation of c-src activity is seen when normal cells are stimulated by growth factors or tumor promoters. The experiments outlined in this proposal focus on the function of pp60c-src in normal growth regulation, and how this function is altered in transformation. We propose to utilize cellular-src specific monoclonal antibodies to measure the tyrosine kinase activity of pp60c-src in cells transformed by several families of retroviruses, DNA tumor viruses and chemical agents as well as from transformed cells which have reverted to the normal phenotype. To determine the molecular basis for the observed alterations in c-src activity from various transformed cells, we will analyze c-src for chemical modifications such as phosphorylations/dephosphorylations, for formation of complexes with viral transforming proteins or cellular regulatory proteins, and for changes in intracellular localization. We also propose to investigate the effect of alterations of c-src activity on its cellular substrates, such as phosphatidyl inositol, and on various cellular target proteins. Similar studies will seek to define and analyze the modulation of c-src in cells treated with growth factors and tumor promoters. To gain insight into the sequence of biochemical events which occur in such responses, we propose to compare the time course of c-src modulation with the time course of activation of growth factor receptors and protein kinase C, and the phosphorylation of various intracellular target proteins. To determine if c-src plays a role in post-membrane events, we will follow its intracellular localization as a function of time after stimulation and test for possible alterations in substrate phosphorylations or substrate specificities. It is the ultimate aim of these studies to help define the function of c-src, particularly as it relates to transmission of growth signals from the cell membrane to the nucleus.
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