Tyrosine kinase oncogenes and receptors regulate cellular activities in part by stimulating the activity of serine/threonine protein kinases. Understanding the flow of regulatory information from the membrane to the cytosol and nucleus thus requires analysis of the mechanisms by which tyrosine kinases communicate with serine/threonine kinases, and of the downstream substrates and functions of these regulated kinases. This proposal is for a molecular, cellular and biochemical analysis of pp42/MAP kinase (termed herein p42mapk) which is a serine/threonine protein kinase regulated by tyrosine and threonine phosphorylations. We take advantage of a full-length P42mapk clone, knowledge of the sites of regulatory phosphorylation and a cell line in which p-42mapk regulation is defective, in order to study the molecular basis of p42mapk regulation and the cellular functions of the enzyme. We will generate regulatory and functionally altered mutants of the enzyme by site-directed mutagenesis. These will be used to study the biochemistry of p42mapk regulation and to identify and characterize regulatory factors which act upstream of the enzyme. We also will use the wild-type and mutant forms of p42mapk to identify and characterize substrates and proteins which bind to the enzyme, and which may play a role downstream from p42mapk in the signaling pathway. P42mapk was first detected as a tyrosine phosphorylated protein in cells transformed by the src oncogene and in growth factor-stimulated cells, but has since been found to become phosphorylated and activated following stimulation with various agonists of secretory, immune and neural cells. Elucidation of the regulation and function of p42mapk should thus be informative about signaling pathways utilized in a variety of cell types and responses, as well as in growth control and malignant transformation.
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