The long-term goal of this project is to understand the functions of TC10, a small GTPase related to Cdc42 and Rac, and of several new effectors for TC10 and Cdc42. A gain-of-function TC10 mutant induces the formation of long peripheral extensions and the loss of stress fibers and membrane ruffles. A two-hybrid screen revealed several new targets that interact with TC10 alone, with both Cdc42 and TC10 or with Cdc42 alone.
The specific aims of the proposal will address the following issues: 1. What are the functions of the Borg family ofTC10/Cdc42-binding proteins? - A new family of 5 proteins related to MSE55 was identified, which have been named Borgs (Binders of Rho-like GTPases). They are involved in actin cytoskeletal remodeling and may inhibit Rho function. Molecular mechanisms underlying these processes, and the functions of endogenous Borg proteins will be identified. 2. What are the functions of the Tribl family of Tc10/Cdc42/Rac-binding proteins? - Tribls are a newfamilyof 4 putative Rho family effectors (TC10/Rac CRIB-like) that contain a partial CRIB-like domain and a PDZ domain. The function of these proteins is unknown. Their role in downstream signaling and binding partners of the PDZ domains will be identified. 3. What is the molecular basis for the differential effects of TC10 and Cdc42 in transformation? -A""""""""rapid-cycling"""""""" mutant of Cdc42, but not of TC10, induces neoplastic transformation in NIH 3T3 cells. Only 3 effectors have been found to interact with Cdc42 but not TC10:WASP, MLK3 and ACK-1. We will determine which of these three effectors is required for Cdc42-mediated transformation. 4. Where and when are TC10 and Cdc42 activated within migrating cells and during cell spreading and cytokinesis? - A novel assay, based on luminescence energy transfer from lanthanide-chelate modified proteins and Cy5-coupled effector proteins will be developed to monitor TC10/Cdc42 activation within living cells. The proposed experiments will uncover important new signaling pathways that regulate cell growth and motility, mediated through the TC10 and Cdc42 GTPases.
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