Cells must be able to sense and respond to a variety of extracellular signals to fulfill their physiological roles. In eukaryotes, chemotaxis and morphogenetic movements in response to extracellular signals control processes such as migration of leukocytes and macrophage in wound healing, cell movements during embryogenesis, axonal guidance, and aggregation of Dictyostelium cells to form a multicellular organism. These diverse processes all require a reorganization of the cytoskeleton that results pseudopod extension and cell migration. Many of the pathways that mediate these changes are highly conserved and use common factors that function in integrated circuits to control directional cell movement toward a chemoattractant. In this application, we focus on two signaling pathways that are involved in controlling cell movement and chemotaxis during the aggregation and multicellular stages of Dictyostelium development. The first is the pathway regulated by the Dictyostelium homologue of Akt/PKB (pkbA), which we have demonstrated is rapidly and transiently activated in response to chemoattractant signals and is essential for cell polarization and movement. In addition, we have identified a second, highly related gene that functions at a slightly later stage in development and may be required for morphogenetic movements necessary for multicellular differentiation. We propose experiments to (1) dissect the mechanisms by which these genes products are activated, (2) characterize cell movement and gene expression defects in strains with null mutations of these genes, and (3) identify downstream effectors of the pathways in which they function. The second pathway uses a MAP kinase activation cascade that plays a central role in integrating extracellular signals and appears to function as a checkpoint for the ability of the cells to respond to chemoattractants. We have demonstrated that the MAP kinase kinase of this cascade, DdMEK1, is essential for cell movement and that it plays a central role in the regulation of receptor activation of guanylyl cyclase, which in turn generates a key second messenger that promotes chemotaxis, and activation of Akt/PKB. Our goals are to (1) identify the other components of the DdMEK1 MAP kinase activation pathway, (2) identify this pathways's downstream effectors and regulators and (3) elucidate the mechanism(s) by which this pathway controls the ability of Dictyostelium cells to respond to the aggregation-stage chemoattractant cAMP. Understanding how the Akt/PKB and DdMEK1 pathways control cell polarization and movement will provide a new understanding of how signaling circuits are integrated to achieve a coherent physiological response.
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