The aim of this investigation is to understand how the phosphorylation of tyrosine hydroxyls in cellular proteins may be involved in the regulation of vertebrate cell growth in culture. Previous experiments by the Principal Investigator and others have established that various mitogens measurable increase the proportion of phosphate that is esterified to proteins via tyrosine. A group of specific cellular proteins, of unknown function, are phosphoylated de novo at tyrosine in response to many mitogens. Some mitogens bind to cell surface receptors that posses tyrosine protien kinase activity, so the proteins we identified may interact directly with these mitogen receptors. Other mitogens whose receptors appear not to be tyrosine protein kinases induce protein phosphorylation by unknown mechanisms. Tyrosine protein kinase activity is also a function of the transforming proteins of some retroviruses, and may be important for the unrestrained proliferation of the cells they transform. It seems likely that since phosphorylation of cell proteins at tyrosine correlates with mitogenesis it is one of several events necessary to trigger proliferation of resting cells. In the first project, a major substrate for mitogen-activated tyrosine protein kinases will be purified, characterized, and used for partial sequence analysis and to immunize animals. A cDNA clone for the protein will be obtained and its sequence derived. The function of the protein in the cell, and its interactions with other cell proteins that may be involved in relay of the mitogenic signal to the nucleus, will be probed using the twin tools of specific antibodies and expression of recombinant DNA clones. In a second project, we will characterize the tyrosine protein kinase(s) that is activated by two classes of mitogens, tumor promoters and mitogenic proteases, whose receptors are not known to possess tyrosine protein kinase activity. This tyrosine protein kinase may be activated following phosphorylation by protien kinase C, the major receptor for tumor promoters in the cell.
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