The goal of this prosal is to understand how signaling pathways invoke changes in the actin cytoskeleton. A number of disease processes are associated with cytoskeletal changes. When cells are ~transformed~ into tumor cells, rearrangements in the actin cytoskeleton increase cell motility and contribute to the changes that allowtumor cells to invade surrounding tissues. Actin also undergoes rapid polymerization when platelets aggregate. Aggregation, which serves to attenuate leakage from bolld vessels, can sometimes restrict blood flow, resulting in strokes and heart attacks. Understanding the regulation of the actin cytoskeleton may provide insighhts into how its reorganization contributes to the progression of a number of important diseases. Many cytoskeletal changes are regulated thorugh the corrdinated actions of several small G proteins including Ras, Rac, Rho and Cdc42. In the budding yeast, Saccharomyces cerevisiae, homologs of the Ras oncogene activate adenylyl cyclase. Ras activation is mediated in vivo through an adenylyl caclase associated protein (CAP). In addition, CAP appears to regulate the actin cytoskeleton through an intrinsic actinmonomer binding activity and through interactions with other cytoskeletal proteins. CAP homologs are also found in mammals. Where tested, all CAP homologs contain the actin cytoskeletal interactions. This proposal aims to test the hypothesis that CAP homologs mediate interactions between the actin cytoskeleton and signaling proteins such as Ras, Rac, Rho and Cdc42. To test this hypothesis we will (1) determine how CAP's interaction with the cytoskeleton regulates Ras/cAMP signaling in yeast, (2) determine the cytoskeletal function of CAP through the isolatioon of CAP-interacting genes in yeast and (3) Purify and characterize CAP from mammalian cells.
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