Endosomal Localization of RacGAP Protein ARHGAP22 Regulates its GAP Activity in Human Melanoma Cells.

Abstract

Rho small GTPases regulate cancer cell adhesion, migration and invasion through reorganization of the actin cytoskeleton. Rho GTPase Activating Protein 22 (ARHGAP22) is a Rac-specific GAP and suppresses Rac-dependent lamella formation and cell spreading. We have previously shown that ARHGAP22 localizes at endosomes in human melanoma A7 cells. The aim of the present study was to demonstrate the functional significance of its localization at the endosomes in melanoma cells. The lamella formation and cell spreading were monitored using human melanoma A7 cells. The effect of inhibition of endosome recycling pathway was examined. We found that dominant negative Rab11 S25N mutant inhibits RacGAP activity of ARHGAP22 and blocks ARHGAP22-dependent suppression of lamella formation and melanoma cell spreading. Furthermore, deletion of 19 amino acid residues at the C-terminal region of ARHGAP22 abolished the localization of ARHGAP22 at enlarged vesicles and stimulated RacGAP activity of ARHGAP22. The deletion mutant accumulated at enlarged vesicles when endosome recycling pathway was blocked either by co-transfection of the Rab11 S25N mutant or treatment of the cells with N-ethylmaleimide, which blocks endosomal vesicular fusion to the plasma membrane. On the other hand, deletion of the pleckstrin homology (PH) domain of ARHGAP22 abolished its RacGAP activity and localization at the plasma membrane. ARHGAP22 localizes at endosomes and is transported to the plasma membrane to inactivate Rac and suppresses lamella formation and spreading of melanoma cells.