Mechanism and Specificity of MAP Kinases

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DESCRIPTION (Applicant's abstract): Mitogen-activated protein kinases (MAPKs) are extremely important enzymes in signal transduction. The consequence of a breakdown in the normal control of these enzymes can lead to many devastating diseases such as cancer. Three major subfamilies have been identified in humans and long-term goals are to identify methods of inhibiting specific members of each subfamily. To achieve this goal a solid chemical-biology approach is taken, where enzymology and molecular biology is combined to establish fundamental properties of the enzymes. The first focus is to define the kinetic mechanism of the extracellular signal-regulated kinase, ERK2, the first MAPK to be discovered, and to test the hypothesis that ADP release is rate-limiting. Sophisticated pre-steady state quench-flow and stopped-flow fluorescence techniques, and equilibrium binding studies, will be used to quantify and identify individual enzymatic steps. The second focus builds on the kinetic model and tests the hypothesis that protein-protein interactions modulate ERK2 activity. A structural analysis-will identify ERK2-substrate interactions and substrate-induced conformational transitions with myelin basic protein and the truncated protein substrates c-Myc(1-100) and Ets-1 (1-138), using trace-labeling experiments. A powerful combination of site-directed mutagenesis and pre-steady state kinetics will critically probe the mechanistic implications of protein-protein interactions mediated by ERK2, focusing on a recently discovered modular binding domain found in many ERK2 substrates. The third focus will use peptide phage display technology to epitope-map ERK2-protein interactions and to test the hypothesis that protein-protein interactions mediated by ERK2 are driven by the recognition of small modular binding sequences that have evolved to form transiently stable complexes. The specific aims during this period are: l) to define the kinetic mechanism of ERK2; 2) to perform structure-function studies to examine ERK2 substrate protein-protein interactions; and 3) to identify tight binding, modular peptide sequences by peptide phage display and contrast their function with wild type modular sequences. This will be the first comprehensive mechanistic study of ERK2 and the discovery of novel peptide inhibitors will set the scene for future structural and cell biology approaches aimed at understanding the breakdown in regulation of one of the primary enzymes in involved in human cancer and disease.