Apoptosis is a program of cellular suicide wherein individual cells are removed from the midst of a living tissue without disturbing overall tissue architecture. The last five years have seen an exponential increase in our understanding of the cellular machinery important for execution of the apoptotic program. However, the signal transduction pathways impacting a cell's decision to live or die are much less well understood. This proposal uses both a cell-free apoptotic system derived from Xenopus eggs and intact mammalian cells to elucidate a signaling pathway involving the adaptor protein Crk and the dual-specificity cell cycle kinase, Wee1. Previous studies demonstrated an interaction between Crk and Wee1 and a requirement for both of these proteins for in vitro apoptotic signaling in egg extracts. Extensions of this work revealed a nuclear export sequence within the Crk protein and an interaction between Crk and Wee1 within the nuclei of mammalian cells. Ablation of the NES increased Crk nuclear accumulation and Crk/Weel interactions, while accelerating apoptotic induction in both mammalian cells and egg extracts. We now propose to determine what role Wee1 plays in Crk-mediated apoptotic signaling and how the Crk/Weel signal is transmitted. Specifically, we will address the role of Wee1 kinase activity/substrates in apoptotic signaling, the importance of Wee1 for Crk nuclear localization, and the recruitment of signaling partners into a Crk/Weel complex. Moreover, we will characterize additional Crk interactors (including those interacting with the Crk N-terminal SH3 domain) to further reconstruct the pathway from Crk to cell death effectors.
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