Colorimetric RhoB GTPase Activity Assay

Background

Although the Rho GTPases RhoA, RhoB, and RhoC share more than 85% amino acid sequence identity, they play distinct roles in tumorigenesis by interacting with different signaling pathways. While the contributions of RhoA and RhoC in tumor development have been detailed in numerous studies, the role of RhoB in cancer progression remains unclear. RhoB controls several fundamental processes such as cell morphology, motility, adhesion, intracellular transport, as well as cell proliferation and mitosis (Spiering and Hodgson, 2011; Ridley et al., 2003; Ju and Gilkes, 2018; Zaoui et al., 2019; Svensmark and Brakebusch, 2019). The role of RhoB in tumorigenesis appears to be complex, as it is likely that RhoB functions in a contextual manner, responding to specific signals in the tumor microenvironment (Mazieres et al., 2004). In addition, RhoB possesses several features that are not found in other Rho GTPases. While the small Rho GTPases are generally localized in the cytoplasm and in response to stimuli, translocate to the plasma membrane, RhoB localizes not only at the plasma membrane and the cytoplasm, but also on endosomes and multivesicular bodies (Wheeler and Ridley, 2004; Vega and Ridley, 2018; Zaoui et al., 2019). In addition, RhoB has a “GCI” (glycine, cysteine, and isoleucine) tripeptide, localized in the hypervariable C-terminus domain (residues 188-190) (Wang and Sebti, 2005). Palmitoylation of cysteine in this tripeptide regulates RhoB stability and RhoB subcellular localization (Perez-Sala et al., 2009). These unique features contribute to the diversity and potentially opposing functions of RhoB in an oncogenic context. In vitro, GTPase assays are extensively used to study general Rho GTPases properties, cycling between active and inactive states, as well as protein expression, and stability. Our studies aim at understanding the role of RhoB GTPase activity on cancer cell signaling regulating cytoskeletal rearrangement, cell migration, and invasion. The Colorimetric RhoB GTPase Activity Assay described herein presents a method to study activation of the RhoB GTPases in these multistep processes, crucial for cancer cell dissemination and metastatic colonization. This methodology may have substantial value in monitoring the level of GTP-loaded RhoB in various contexts such as fundamental research on cell motility as well as therapeutic compounds development.