Abstract
The small GTP-binding protein Arf6 reorganizes the actin cytoskeleton through the regulation of Rac activity. We identified FilGAP, a Rac-specific Rho GTPase-activating protein that is recruited to plasma membranes by binding to activated Arf6. FilGAP binds to Arf6 through its pleckstrin homology domain. Activated Arf6 stimulated RacGAP activity of FilGAP, and knockdown of endogenous Arf6 by siRNA suppresses FilGAP-mediated bleb formation. Mutant FilGAP lacking phosphatidylinositol 3,4,5-trisphosphate (PIP3) binding (FilGAP R39C) binds to activated Arf6 and induces bleb formation. Moreover, bleb formation induced by wild-type FilGAP occurs in the presence of phosphatidylinositol 3-kinase inhibitors, suggesting a PIP3-independent interaction between FilGAP and Arf6. We propose that FilGAP may function as a mediator of the regulation of Rac by Arf6.
Highlights
FilGAP is a Rac GTPase-activating protein, but how it is regulated remains unclear
RhoGAP-deficient mutant FilGAP R175A was engineered on the basis of the observation that RhoGAPs have key arginine residues that mediate their catalytic activity and that arginine at position 175 is the relevant residue in FilGAP [11, 21]
Arf6 controls actin remodeling, cell shape change, and cell migration, and many of the Arf6-dependent regulation of the actin cytoskeleton is mediated by Rac [2,3,4]
Summary
Results: GTPase Arf binds to FilGAP and stimulates its RacGAP activity to induce plasma membrane blebbing. Significance: This study establishes a novel molecular link between Arf and FilGAP that may have a role in Arf6-dependent inactivation of Rac. The small GTP-binding protein Arf reorganizes the actin cytoskeleton through the regulation of Rac activity. We identified FilGAP, a Rac-specific Rho GTPase-activating protein that is recruited to plasma membranes by binding to activated Arf. Mutant FilGAP lacking phosphatidylinositol 3,4,5-trisphosphate (PIP3) binding (FilGAP R39C) binds to activated Arf and induces bleb formation. FilGAP is a Rho GTPase-activating protein (GAP) and binds to the actin filament cross-linking protein filamin A (FLNa) [11,12,13,14,15,16,17]. FilGAP may inactivate Rac as a downstream effector of Arf
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