When cells become transformed by Rous sarcoma virus (RSV) they undergo numerous biochemical and regulatory changes which collectively constitute the """"""""transformed phenotype."""""""" All the manifestations of the transformed phenotype are dependent on the continuous activity of the viral transforming protein, pp60src, which has a tyrosine-specific protein kinase actvity. Genetic evidence indicates that pp60src has severa physiologically significant cellular targets with which it interacts to generate the transformed phenotype, and direct biochemical analysis reveals that numerous proteins become phosphorylated on tyrosine during transformatin by RSV. However, it is not know which of the tyrosine-phosphorylated proteins are physiologically significant targets nor what role these phosphorylations play in the genesis of the transformed phenotype. In this application, we propose a biochemical and genetic analysis of the intermediate steps leading from tyrosine phosphorylation by pp60src to the transformed phenotype. to simplify this analysis we will focus on biochemical and regulatory events which are shared by mitogen-treated and RSV-transformed cells: 1. We will isolate cellular variants which are altered in their response to pp60src or mitogenic agents. Refractoriness to either increased growth or increased glucose transport will be used as the basis for selection. Such variants may carry mutations in genes coding for targets of pp60src. 2. We will purify and characterize selected phosphotyrosine-containing proteins, with the first priority being to purify a protein of 42,000 Mr which is phosphorylated in both mitogen-stimulated cells and in RSV-transformed cells. The purified proteins will be used to raise monoclonal antibodies or antisera for studies on localization, for micro-injection and for direct functional studies. By taking the approach of biochemical genetics, we hope to define some of the intermediate steps in transformation by RSV and particularly the role of specific tyrosine phosphorylations in growth control and malignant transformation.
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