Rho-like GTPases such as rhoA, rac1, and cdc42 play an essential role in growth factor-mediated changes in the actin cytoskeleton necessary for cell growth and motility. These GTPases are activated by guanine nucleotide exchange factors (GEFs) which promote the exchange of GDP for GTP, and are negatively regulated by GTPases activating proteins. The multifunctional Trio protein contains a rac1-specific GEF domain, a rhoA-specific GEF domain, a protein serine threonine kinase domain, and a number of auxiliary domains. Expression of the Trio rac1 GEF domain causes membrane ruffling and expression of the rhoA Trio GEF domain causes increased cell-matrix interactions via focal adhesion formation. Expression of the Trio rac1-specific GEF domain also causes anchorage independent growth of NIH 3T3 cells indicating that Trio is a potential proto-oncogene. Trio binds the focal adhesion-associated LAR transmembrane protein tyrosine phosphatase, suggesting that a Trio-LAR complex could also regulate protein tyrosine phosphorylation signaling. Given the central role of cell migration in metastasis, Trio may be particularly important in human cancer, as aberrant Trio expression may dysregulate cell growth and/or cell motility. Thus, we will study the Trio protein to understand how external signals are transmitted into the cell and how these signals lead to changes in cell growth and cell motility. The overall aim of this proposal is to test our hypothesis that Trio is an integrator/broadcaster of intracellular signal flow that coordinates multiple signals to regulate cytoskeletal changes associated with cell growth and cell motility.
The specific aims are: 1)to identify signaling events regulating Trio functions, 2) determine the role of the three Trio enzymatic domains within context of the entire Trio protein, and 3) to analyze Trio signal transduction via the isolation of Trio binding proteins. The health-relatedness of this project is to increase our understanding of the signaling pathways that regulate cell growth and motility. Malfunction of such mechanisms could result in cell transformation and/or tumor metastasis.
The aims will be accomplished by: i) identifying what upstream signals regulate Trio phosphorylation and localization, and determining how altered phosphorylation and/or localization affects Trio enzymatic activities; ii) determining what roles Trio plays in regulating cell morphology, motility and growth; and iii) identifying and characterizing Trio binding proteins, and determining the effect of the Trio-binding proteins on Trio signaling.
