The long-term goal of this research program is to elucidate the cellular and molecular mechanisms by which the polypeptide hormone epidermal growth factor (EGF) stimulates eukaryotic cell replication. Cellular protein-tyrosine kinase activity is stimulated by EGF and the oncogenes of a number of tumor viruses code for protein-tyrosine kinase. This and other parallels between EGF-stimulated growth and the rapid growth associated with cells transformed by tumor viruses make it reasonable to propose that EGF stimulates cell replication by stimulating protein-tyrosine kinases that phosphorylate, and thus modulate the activity of, growth regulatory proteins. Experiments are proposed that will purify and characterize substrates that have the characteristics of such regulatory proteins. In other experiments EGF-stimulated protein kinases will be investigated. Although it is clear that the EGF receptor is itself a kinase, preliminary evidence is presented which identifies another protein-tyrosine kinase that can be stimulated by allosteric interaction with the occupied EGF receptor. A primary goal of this proposal is to purify this kinase and reconstitute in vitro an EGF stimulation of purified substrate phosphorylation using the purified kinase. The kinase and substrate purifications will use placenta as the starting material because it is a normal, highly proliferative tissue that can be obtained in large amounts. The primary amino acid sequence of the purified kinase and substrate will be determined by molecular cloning and sequence analysis of the cloned cDNA. Probes to the purified proteins will be prepared and used to begin investigations of the physiological function of this kinase and substrate in intact cultured cells that have well- characterized mitogenic responses to EGF.
