Divergence of Rho residue 43 impacts GEF activity.
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.
Department of Chemistry, Wartburg College; Waverly, IA USA.
Christina M
CM
Sloan
Clancy V
CV
Quinn
Justin P
JP
Peters
Janean
J
Farley
Chris
C
Goetzinger
Molly
M
Wernli
Kris A
KA
DeMali
Shawn M
SM
Ellerbroek
D000818:Animals; D045744:Cell Line, Tumor; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D051379:Mice; D041681:NIH 3T3 Cells; D017354:Point Mutation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020741:rho GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020742:rhoA GTP-Binding Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020743:rhoB GTP-Binding Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000075024:rhoC GTP-Binding Protein
D020662:Guanine Nucleotide Exchange Factors; D011993:Recombinant Fusion Proteins; D006160:Guanosine Triphosphate; C066690:RHOC protein, human; D020741:rho GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein; D020743:rhoB GTP-Binding Protein; D000075024:rhoC GTP-Binding Protein