Real-time NMR study of guanine nucleotide exchange and activation of RhoA by PDZ-RhoGEF.
Small guanosine triphosphatases (GTPases) become activated when GDP is replaced by GTP at the highly conserved nucleotide binding site. This process is intrinsically very slow in most GTPases but is significantly accelerated by guanine nucleotide exchange factors (GEFs). Nucleotide exchange in small GTPases has been widely studied using spectroscopy with fluorescently tagged nucleotides. However, this method suffers from effects of the bulky fluorescent moiety covalently attached to the nucleotide. Here, we have used a newly developed real-time NMR-based assay to monitor small GTPase RhoA nucleotide exchange by probing the RhoA conformation. We compared RhoA nucleotide exchange from GDP to GTP and GTP analogues in the absence and presence of the catalytic DH-PH domain of PDZ-RhoGEF (DH-PH(PRG)). Using the non-hydrolyzable analogue guanosine-5'-O-(3-thiotriphosphate), which we found to be a reliable mimic of GTP, we obtained an intrinsic nucleotide exchange rate of 5.5 x 10(-4) min(-1). This reaction is markedly accelerated to 1179 x 10(-4) min(-1) in the presence of DH-PH(PRG) at a ratio of 1:8,000 relative to RhoA. Mutagenesis studies confirmed the importance of Arg-868 near a conserved region (CR3) of the Dbl homology (DH) domain and revealed that Glu-741 in CR1 is critical for full activity of DH-PH(PRG), together suggesting that the catalytic mechanism of PDZ-RhoGEF is similar to Tiam1. Mutation of the single RhoA (E97A) residue that contacts the pleckstrin homology (PH) domain rendered the mutant 10-fold less sensitive to the activity of DH-PH(PRG). Interestingly, this mutation does not affect RhoA activation by leukemia-associated RhoGEF (LARG), indicating that the PH domains of these two homologous GEFs may play different roles.
Gasmi-Seabrook
Geneviève M C
GM
Division of Signaling Biology, Ontario Cancer Institute, University Health Network, Toronto, Ontario M5G 1L7, Canada.
Marshall
Christopher B
CB
Cheung
Melissa
M
Kim
Bryan
B
Wang
Feng
F
Jang
Ying Ju
YJ
Mak
Tak W
TW
Stambolic
Vuk
V
Ikura
Mitsuhiko
M
D019943:Amino Acid Substitution; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000737:chemistry* / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D016296:Mutagenesis; D020125:Mutation, Missense; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D017434:Protein Structure, Tertiary; D064067:Rho Guanine Nucleotide Exchange Factors; D020742:rhoA GTP-Binding Protein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
C577222:ARHGEF12 protein, human; D020662:Guanine Nucleotide Exchange Factors; D064067:Rho Guanine Nucleotide Exchange Factors; C066737:RHOA protein, human; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D020742:rhoA GTP-Binding Protein