An Arf1 GTPase mutant with different responses to GEF inhibitors |
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Authors: | Flisiak Sebastian Zeeh Jean-Christophe Guibert Bernard Cherfils Jacqueline Zeghouf Mahel |
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Affiliation: | Laboratoire d’Enzymologie et Biochimie Structurales (LEBS), CNRS, Avenue de la Terrasse, 91198 Gif-sur-Yvette, France |
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Abstract: | 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. |
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Keywords: | GTPase Arf ADP ribosylation factor Guanine nucleotide exchange factor GEF Sec7 ARNO Protein-protein interactions Inhibitor Fluorescence kinetics |
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