Rho small GTPases control cell morphology and motility through the rearrangement of actin cytoskeleton. We have previously shown that FilGAP, a Rac-specific GAP, binds to the actin-cross-linking protein Filamin A (FLNa) and suppresses Rac-dependent lamellae formation and cell spreading. ARHGAP22 is a member of FilGAP family, and implicated in the regulation of tumor cell motility. However, little is known concerning the cellular localization and mechanism of regulation at the molecular level. Whereas FilGAP binds to FLNa and localizes to lamellae, we found that ARHGAP22 did not bind to FLNa. Forced expression of ARHGAP22 induced enlarged vesicular structures containing the endocytic markers EEA1, Rab5, and Rab11. Moreover, endogenous ARHGAP22 is co-localized with EEA1- and Rab11-positive endosomes but not with trans-Golgi marker TNG46. When constitutively activated Rac Q61L mutant was expressed, ARHGAP22 is co-localized with Rac Q61L at membrane ruffles, suggesting that ARHGAP22 is translocated from endosomes to membrane ruffles to inactivate Rac. Forced expression of ARHGAP22 suppressed lamellae formation and cell spreading. Conversely, knockdown of endogenous ARHGAP22 stimulated cell spreading. Thus, our findings suggest that ARHGAP22 controls cell morphology by inactivating Rac but its localization is not mediated by its interaction with FLNa.

Malignant tumor cells acquire abnormal motility and invade through various extracellular matrix (ECM) components with distinct composition and architecture, including basement membrane and interstitial collagen networks, and leave a blood or lymphatic vessel and invade the surrounding tissue parenchyma. 1,2 Actin cytoskeletal dynamics is essential for cell polarization and migration. 3-6 Migrating cells are characterized by a sustained front-rear asymmetry, with a front enriched in filamentous actin (F-actin) and a rear made of actomyosin filaments. Rho small GTPase (Rho GTPase) Rac1 induces membrane protrusion at the front of migrating cells through actin polymerization, whereas the activity of another Rho GTPase RhoA is required for tail retraction at the rear through activation of actomyosin contraction. 3,7-10 This acquisition of front-rear polarity is important for effective motility of various cell types including tumor cells.

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