Chemotaxis, or directed cell movement toward a small molecule ligand, plays a key role in many cellular and physiological responses, including metastasis of cancer cells, movement of neutrophils and macrophage in immunity, migration of embryonic cells during development, and aggregation of Dictyostelium during development. Cells must be able to respond to a shallow extracellular chemoattractant gradient and convert this into a steep intracellular gradient of signaling components, which controls the spatially restricted polymerization of F-actin at the leading edge (and to a lesser degree the cell's posterior) and assembly and contraction of myosin II at the cell's posterior. This requires the integration of several signal transduction pathways, many of which are conserved between Dictyostelium and man. Recent findings have established that phosphatidylinositol 3-kinase (PI3K) and MAP kinases cascades as key regulators of a cell's responses to directional signals. This proposal focuses on the further analysis of the Dictyostelium MEK1/ERK1 MAP kinase cascade and its role in controlling chemotaxis. The proposal takes advantage of the biochemical, genetic, and cell biological approaches available to dissect regulatory pathways in this experimental system. We have demonstrated that MEK1 and its downstream MAP kinase ERK1 are required for proper chemotaxis and that both components localize to the leading edge in chemotaxing cells. We have discovered that the control of the subcellular localization of these components requires the reversible SUMOylation of MEK1 and that adaptation of this pathway, which is essential for proper development, involves feedback regulation by the ERK1 and PI3K pathways. Our goal is to elucidate how MEK1 and ERK1 regulate chemotaxis by determining which chemotaxis functions are defective in mek1/erk1 null cells. We will identify and examine the function of ERK1 substrates through two-hybrid screens and then examine their function through biochemical approaches, and mutant screens. In addition, we will elucidate the mechanisms that control MEK1 deSUMOylation and pathway adaptation. Lastly, we will determine the role of SMEK1, an evolutionarily-conserved, second-site suppressor of MEK1 that was identified in my lab in regulating the MEK 1/ERK1 pathway and chemotaxis. The work proposed in this application should provide new and important insights into mechanisms that control this highly evolutionarily conserved cell biological process, and thus provide the needed background to elucidate the cellular basis underlying a variety of human diseases, including those affecting innate immunity and metastasis of cancer cells.
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