| Rho GTPase signaling pathways play critical roles in both normal cellular biology and cancer biology. They appear to be involved in all stages of tumorigenesis including dedifferentiation, uncontrolled proliferation, angiogenesis, invasion and metastasis. As a member of this family of proteins, Rac1 GTPase functions as a molecular switch which cycles between an active GTP-bound and an inactive GDP-bound state. When in the active state, it is able to interact with effector proteins leading to crucial biological outcomes such as cytoskeleton reorganization, cell migration, and cell cycle progression. Rac1b, a splice variant of Rac1, is a self-activating protein with a rapid GDP/GTP exchange and impaired GTP hydrolysis. With these properties, Rac1b exists in an active GTP bound state in the cell and is predominantly localized to the plasma membrane. Rac1b has been shown to be upregulated in colon carcinoma; however the function of this protein is not yet understood.;The goal of this dissertation is to characterize the properties and to determine the functional significance of Rac1b. In this study, we have made the striking discovery that Rac1b negatively regulates its splice variant Rac1. We show that Rac1b decreases the levels of activated Rac1 following growth factor stimulation. As a consequence, it negatively impacts on Rac1 downstream signaling events. We show that the mechanism of Rac1 inhibition is by Rac1b interfering with Rac1 membrane recruitment in response to upstream signaling. As a result, Rac1b inhibits Rac1-dependent cytoskeleton rearrangement and cell morphology, and enhances invasive behavior of colorectal cancer cells. These findings provide a novel function of Rac1b as a negative regulator of Rac1 and demonstrate a novel mechanism by which Rac1 is negatively regulated in the cell. This is the first time that negative regulation of a small GTPase by its splice variant has been described. |
