Divergence of Rho residue 43 impacts GEF activity

• Published in: Small GTPases Vol. 3; no. 1; pp. 15 - 22
• Main Authors: Sloan, Christina M., Quinn, Clancy V., Peters, Justin P., Farley, Janean, Goetzinger, Chris, Wernli, Molly, DeMali, Kris A., Ellerbroek, Shawn M.
• Format: Journal Article
• Language: English
• Published: United States Taylor & Francis 01.01.2012; Landes Bioscience
• Subjects: Animals; Binding; Biology; Bioscience; Calcium; Cancer; Cell; Cell Line, Tumor; Cycle; GEF; Guanine Nucleotide Exchange Factors - metabolism; Guanosine Triphosphate - metabolism; Humans; Landes; Mice; NIH 3T3 Cells; nucleotide exchange; Organogenesis; Point Mutation; Proteins; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Research Paper; Residue 43; RHO; rho GTP-Binding Proteins - chemistry; rho GTP-Binding Proteins - genetics; rho GTP-Binding Proteins - metabolism; rhoA GTP-Binding Protein - chemistry; rhoA GTP-Binding Protein - genetics; rhoA GTP-Binding Protein - metabolism; rhoB GTP-Binding Protein - chemistry; rhoB GTP-Binding Protein - genetics; rhoB GTP-Binding Protein - metabolism; rhoC GTP-Binding Protein
• ISSN: 2154-1248; 2154-1256
• DOI: 10.4161/sgtp.19557

RhoA, RhoB and RhoC GTPases are over 85% identical at the amino acid level, with RhoA and RhoC differing at only one residue (43) across the initial two-thirds of their sequences. A putative regulatory distinction between the molecules is their capacity to be uniquely activated by guanine nucleotide exchange factors (GEFs). We hypothesize that variation of amino acid residue 43 between RhoA/B (valine) and RhoC (isoleucine) impacts GEF activity. Direct participation of residue 43 in GEF-catalyzed exchange was confirmed by the observation that mutation of this position to a threonine reduced GEF-catalyzed nucleotide exchange activity in vitro (Vav2, XPLN, GEFT, Dbl and Dbs) and greatly depressed RhoA and RhoC GTP-loading profiles in cell lysates. Using a residue swap approach, substitution of RhoA Val 43 with an Ile was found to significantly promote basal nucleotide exchange activity and enhance GTP-loading in cells. Substitution of Val 43 with an Ile in RhoB negatively affected nucleotide exchange in vitro. Substitution of RhoC Ile 43 with a Val increased GEF-catalyzed exchange in vitro. In addition, RhoC-I43V was more efficacious at driving ovarian cancer cell invasion through matrigrel than wild-type RhoC, RhoC-I43T, wild-type RhoA, RhoA-V43I or RhoA-V43T GTPases. These findings suggest that a divergence between RhoA/B and RhoC at residue 43 impacts basal and GEF-stimulated nucleotide exchange activity.