Protein phosphorylation is a primary mechanism for controlling the activity of enzymes and other proteins. This regulatory mechanism is involved in the control of a wide variety of cellular processes. Recently, a novel class of protein kinase has been identified which phosphorylates target proteins on tyrosine residues rather than the more common serine or threonine residues. These tyrosine phosphorylation reactions have been implicated in the control of cell growth in normal cells and in cells transformed by RNA tumor viruses. The steady state level of phosphorylation of any cellular protein is determined by the balance of the activities of the protein kinases and protein phosphatases acting on the protein. Four enzymes, termed protein phosphatases 1, 2A, 2B and 2C, have been shown to be the major protein phosphatases acting on phosphoseryl and phosphothreonyl proteins involved in regulating intermediary metabolism. It is now clear that these protein phosphatases are important targets for cellular regulation and specific mechanisms have been identified for their control by cAMP, calcium ions and calmodulin. Little is presently known about the properties and mechanisms for regulating the phosphotyrosyl protein phosphatases. However, in preliminary experiments, five species of phosphotyrosyl protein phosphatase were resolved by successive chromatographies of bovine brain extracts on DEAE-cellulose and phosphocellulose, using phosphotyrosyl casein as a model substrate. At least three of these species, termed PC2, PC4 and PC5, appear to be quite distinct from protein phosphatases 1, 2A, 2B and 2C. These three species represent the major phosphotyrosyl casein phosphatase activities in brain extracts. The goals of the proposed project are to purify and characterize PC2, PC4 and PC5 from bovine brain and to develop antibodies to each of them. Peptide mapping techniques and a comparison of antibody crossreactivities will then be used to determine whether the five species of phosphotyrosyl casein phosphatase are related to one another or to protein phosphatases 1, 2A, 2B and 2C. Experiments will also be carried out to determine whether mechanisms exist for regulating PC2, PC4 and PC5. Finally, two proteins (i.e. pp60src and p34) known to be phosphorylated on tyrosine residues in vivo will be used to assess the physiological relevance of the phosphotyrosyl casein phosphatases. The proposed studies should provide important insights into the role of tyrosine protein phosphorylation in the regulation of cell growth and in oncogenesis.
