An Arf1 GTPase mutant with different responses to GEF inhibitors.
Guanine nucleotide exchange factors (GEFs) stimulate the activation of small GTP-binding proteins (GTPases). Establishing their specificity is a challenging issue, in which chemical genetics are rapidly gaining interest. We report a mutation in the Arf1 GTPase, K38A, which differentially alters its sensitivity to GEF inhibitors. The mutation renders Arf1 insensitive to LM11, a GEF inhibitor that we previously discovered by structure-based screening. In contrast, full inhibition by the natural compound Brefeldin A (BFA) is retained. We show that the mutation is otherwise silent towards the biochemical and cellular properties of Arf1, notably its binding to effectors as measured by a novel GEF-protection assay. This is thus the first GTPase mutant with different responses to two classes of inhibitors, and a novel tool to analyze Arf and ArfGEF specificity and functions in vitro and in cells.
Laboratoire d'Enzymologie et Biochimie Structurales, CNRS, Gif-sur-Yvette, France.
Sebastian
S
Flisiak
Jean-Christophe
JC
Zeeh
Bernard
B
Guibert
Jacqueline
J
Cherfils
Mahel
M
Zeghouf
D020823:ADP-Ribosylation Factor 1 / Q000737:chemistry / Q000187:drug effects* / Q000235:genetics*; D000409:Alanine / Q000737:chemistry / Q000235:genetics; D000814:Aniline Compounds / Q000494:pharmacology*; D001562:Benzimidazoles / Q000494:pharmacology*; D020126:Brefeldin A / Q000494:pharmacology*; D018360:Crystallography, X-Ray; D020690:GTPase-Activating Proteins / Q000737:chemistry / Q000187:drug effects / Q000235:genetics; D020662:Guanine Nucleotide Exchange Factors / Q000037:antagonists & inhibitors; D006367:HeLa Cells; D006801:Humans; D008239:Lysine / Q000737:chemistry / Q000235:genetics; D009154:Mutation; D011487:Protein Conformation
D000814:Aniline Compounds; D001562:Benzimidazoles; D020690:GTPase-Activating Proteins; D020662:Guanine Nucleotide Exchange Factors; C521938:LM11 compound; C102474:cytohesin-2; D020126:Brefeldin A; D020823:ADP-Ribosylation Factor 1; D008239:Lysine; D000409:Alanine