Chemical Genetics as a Tool to Dissect the Regulation of Rho GTPase Signaling

Small-molecule inhibitors can be used to dissect complex cellular signaling networks based on their ability to rapidly inhibit individual proteins participating within a network. For example, kinase inhibitors have been invaluable reagents for revealing epistatic relationships in signal transduction cascades (Knight and Shokat 2005). An example of a complex signaling network whose study could benefit from specific inhibitors is the signaling network centered around the Rho subfamily of the Ras superfamily of small GTP-binding proteins. The Rho GTPases are a family of signaling proteins originally linked closely to the regulation of the actin cytoskeleton (Ridley and Hall 1992; Ridley et al. 1992; Nobes and Hall 1995); however, ensuing studies have uncovered a myriad of cellular signaling pathways that involve the Rho GTPases (Jaffe and Hall 2005). As such, Rho GTPases have been implicated in a plethora of biological functions and disease states, including membrane trafficking, mitosis, transformation, and cancer (reviewed in Sahai and Marshall 2002).

The complexity of Rho GTPase signaling is due largely to the extensive number of both upstream and downstream binding partners. Rho proteins are molecular switches that cycle between inactive GDP-bound and active GTP-bound states through the actions of upstream regulatory proteins. GTPase-activating proteins promote the hydrolysis of bound GTP to GDP, and guanine nucleotide exchange factors catalyze the release of bound GDP, facilitating binding of GTP (Bos et al. 2007). Over 70 guanine nucleotide exchange factors have been identified for Rho GTPases (Meller et al. 2005; Rossman et al. 2005; Bos et al. 2007), thereby providing a mechanism for coupling a broad spectrum of upstream pathways to Rho GTPase activation. When GTP-bound, Rho GTPases can bind any of over 30 different effector proteins, including enzymes (e.g., kinases, oxidases, and lipases) as well as proteins that appear to serve predominantly scaffolding functions (Karnoub et al. 2004). However, the biological roles of many of these effectors are poorly understood, and a major emphasis in the field of Rho GTPase signaling has been the identification and functional characterization of upstream and downstream binding partners.

Adding to the complexity is the existence of 20 different Rho GTPases in humans (Boureux et al. 2007). Some binding partners are shared by multiple Rho GTPases, whereas others appear more GTPase-specific (Karnoub et al. 2004). Three of the best-studied Rho GTPases are Rho, Rac, and Cdc42. Rac and Cdc42 are the most closely related and share a number of effector proteins (Karnoub et al. 2004). The Rho family, by contrast, signals via a largely non-overlapping series of effectors (Karnoub et al. 2004).

The development of small-molecule inhibitors of components of Rho GTPase-mediated signaling could facilitate the elucidation of the functional roles of the proteins within these networks and may substantially further our ability to define the nature of the complex signaling networks themselves. In the following section, we will describe a forward chemical genetic screen to identify small molecules that may target proteins both upstream and downstream of Cdc42, a Rho GTPase identified as a key signaling module in the regulation of actin filament assembly.

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