Abnormal induction of apoptosis plays an important role in a wide range of disease, including autoimmune disorders, malignancies, and degenerative diseases, Preliminary studies have shown that the Rho family of small GTPases have a profound effect on apoptosis induction by both cytotoxic T cells and Fas. These studies have led us to postulate the existence of a novel signaling cascade controlling apoptosis, in which PI3 kinase activates Rho proteins, which in turn produce cytoskeletal changes through elevation of PIP2. In the studies proposed here, the existence of this pathway will be confirmed, the sequence of interactions will be determined, and the involvement of signaling between Rho family members will be investigated. Combinations of specific inhibitors, and active and inhibitory mutants in the same cell will e used to dissect out the sequence of signaling steps. The role of the cytoskeleton and subcellular localization of GTPases will be examined. Because membrane localization is important in controlling the downstream effects of the GTPases,. The sequence and extent of membrane translocation will be quantified for each GTPase during activation, using fluorescence imaging of GFP fusion proteins in living cells. Dynamics of focal adhesions and their control by Rho family proteins in apoptosis will be investigated. The studies proposed here will elucidate a previously unknown pathway controlling apoptosis through ubiquitous and highly conserved proteins.
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