Activated receptor and nonreceptor protein tyrosine kinases (PTKs) are presumed to mediate their diverse effects in signal transduction and cell transformation by phosphorylating cellular proteins on tyrosine and thereby modifying their biochemical activities. Because many cellular substrates of PTKs have not been molecularly characterized and their functions remain unknown, our understanding of the biochemical pathways that relay PTK- induced signals remains incomplete. The central theme of this proposal is to characterize novel PTK substrates and to determine their putative role(s) in signal transduction and cell transformation. Using monoclonal antibodies prepared to different proteins phosphorylated by the src tyrosine kinase, I have recently cloned cDNAs encoding eight cellular substrates, seven of which show no overall homology to sequences in international protein databases. I plan to focus on the characterization of a representative 120 kilodalton (kDa) protein (p120) whose phosphorylation in cells transformed by activated p60c-src, polyoma middle T antigen, and in response to stimulation of cells by epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and colony-stimulating factor 1 (CSF-1) implies that it may play a central role in ligand-induced signaling and cell transformation. My goals are to complete the molecular characterization of p120 cDNA, to determine its subcellular topology and distribution among different cell types, and to biochemically map the major sites of tyrosine phosphorylation within the molecule. Using this information, I hope to develop model biological systems for defining the as yet unknown function of p120 and for determining the role of tyrosine phosphorylation in modifying its activity. In principle, the approaches that I envision should be applicable to the characterization of the other src substrates defined by my antibodies, and I will include experiments involving these proteins under circumstances where their comparison with p120 might prove particularly informative.
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