The long term goals of this project are to dissect the unique as well as the commonly shared structure-function properties of individual members of Rho family GTPases and to apply the mechanistic knowledge of specific regulatory and effector interactions of Rho GTPases to the design of biochemical reagents targeting at Rho protein functions. The Rho family GTPases RhoA, Rac1, and Cdc42 are molecular switches that control a wide variety of signal transduction pathways regulating cell actin cytoskeletal organization, microtubule dynamics, transcriptional activity, membrane trafficking and cell cycle progression. Aberrant signaling through Rho proteins may lead to certain human diseases such as cancer and inflammation. Three classes of proteins that control their GTPase cycle, i.e. the guanine nucleotide exchange factors (GEFs), GTPase-activating proteins (GAPs), and the guanine nucleotide dissociation inhibitors (GDIs), are involved in regulating their cellular activities. In addition, a large array of putative effectors that may serve to mediate their physiological responses has also been identified. Building on our previous structure-function studies of Rho GTPases, we propose three specific aims focusing on Rac1 regulation and function in the current studies.
In aim 1, we will examine the mechanism of interaction of Rac1 GTPase with a few novel regulatory GEF/GAP proteins and effector targets by a mutagenesis approach and study Rac1 regulation and signaling mechanisms in Rac1 knockout cells.
In aim 2, we will reconstitute the GTP-binding/GTP- hydrolysis cycle of Rac1 GTPase in vitro and determine the effect of possible molecular interplay of various Rac1 regulators and effectors on the effector activation.
In aim 3, we will utilize the mechanistic knowledge of Rac-GEF interaction to characterize a small molecule inhibitor targeting at the specific interaction of Rac1 with its GEFs and test its anti-tumorigenic potential in a variety of cancer cells in which Rac GTPase signaling is altered. These studies will provide mechanistic insights into the complex regulatory and effector interactions of Rho GTPases and may generate pharmacological reagents that interfere with specific functions of Rho GTPases contributing to human diseases.
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