Mechanisms of transduction of growth signals in normal and malignant cells are becoming clearer through analysis of cytoplasmic kinases that are activated by these signals. Stimulation of this kinase cascade is a frequent consequence, and possible also a cause, of cancer formation. A central target of this cascade appears to the MAP kinase, since MAP kinase is activated via numerous mitogenic stimuli including those that are perturbed in oncogenic transformation. Current evidence is that MAP kinase is activated by phosphorylation by another kinase, termed MEK, and that MEK can be activated by two distinct kinases, raf and MEK kinase. Unanswered is the question of how these individual kinases regulate cell transformation and other growth-related processes. To date, genetic tools that would be of use to characterize the role of MEK in the MAP kinase activation pathway have not been described. We propose to investigate MEK physiology in molecular detail, and use this information to develop active or inhibitory mutants of MEK to test the hypothesis that MEK participates in cellular processes vital for cell transformation, transcriptional stimulation, and other features of growth stimulation. Therefore, we propose: 1) To identify sites of phosphorylation on MEK by raf and MEK kinase. 2) To identify and characterize constitutive active mutants of MEK that do not require phosphorylation for activity. 3) To identify and characterize dominant inhibitory mutants of MEK and to use them to characterize signal transduction through MEK. 4) To identify sites of phosphorylation on MEK by MAP kinase. (Such """"""""feedback"""""""" phosphorylation events may result in autoregulation of MAP kinase activity via an autoregulatory loop). 5) To test MEK kinase activation following truncation of a regulatory domain, and to measure the ability of MEK kinase to funcTIon as an oncogene.
