This grant concerns aspects of cytoskeletal organization, cell cycle regulation, and signal transduction. The major approaches are biochemical and biophysical and the starting point for most of the experiments is the capacity to recreate complex processes in concentrated Xenopus egg extracts in vitro. We have recently investigated the control of actin nucleation by phosphoinositides and small GTP binding proteins. This has led to the reconstitution of a process involving the proteins N-WASP, Cdc42, the Arp 2,3 complex and actin and the lipid, phosphoinositol (2,5) bisposphate, underlying actin assembly in cells. We propose to identify new pathways for actin regulation and explore the exact mechanism by which these components regulate actin nucleation. We plan to study the activation of the Arp 2,3 complex, crystallize it and determine its structure at the atomic level. We plan to use the extract system or the purified components to screen for new synthetic inhibitors of actin nucleation, screening against very diverse libraries of combinatorically synthesized, natural product-like molecules. We plan to investigate the putative conformational change in the mitotic activator, Cdc25, induced by the prolyl isomerase Pin-1 and we plan to study the interaction of the phosposeryl/ phosphothreonyl binding proteins 14-3-3 with Pin-1 in their binding to Cdc25. Finally we plan to study the newly identified vertebrate form of the general anaphase inhibitor, Pds1, to determine its biochemical activities and the activities of its partner, ESP1 in causing chromatid separation at anaphase. Specifically we will also be looking for clues for its role in generating aneuploidy and the reasons why it is oncogenic.
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