RESUMEN
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.
Asunto(s)
Factores de Ribosilacion-ADP/metabolismo , Membrana Celular/metabolismo , Regulación hacia Abajo/fisiología , Proteínas Activadoras de GTPasa/metabolismo , Proteína de Unión al GTP rac1/biosíntesis , Factor 6 de Ribosilación del ADP , Factores de Ribosilacion-ADP/genética , Línea Celular Tumoral , Membrana Celular/genética , Proteínas Activadoras de GTPasa/genética , Células HEK293 , Humanos , Fosfatos de Fosfatidilinositol/genética , Fosfatos de Fosfatidilinositol/metabolismo , Proteína de Unión al GTP rac1/genéticaRESUMEN
BACKGROUND/AIM: The small GTPase ADP ribosylation factor 6 (ARF6) promotes carcinoma cell invasion and metastasis through remodeling of actin cytoskeleton and formation of pseudopod that is regulated by RAC. RHO GTPase activating protein 24 (ARHGAP24), a RAC-specific GTPase activating protein, binds to activated ARF6 and is recruited to the plasma membrane. The aim of the present study was to demonstrate if ARHGAP24 is involved in the ARF6-mediated formation of pseudopods in breast carcinoma cells. MATERIALS AND METHODS: The formation of pseudopods induced by activated ARF6 was monitored using MDA-MB-231 human breast carcinoma cells. The effect of knockdown of endogenous ARHGAP24 by siRNA was examined. RESULTS: Knockdown of ARHGAP24 in MDA-MB-231 carcinoma cells increased the lifespan of pseudopods to retract, which resulted in increased length of pseudopods induced by activated ARF6. ARHGAP24 required a binding site of ARF6 to achieve ARF6-dependent actin remodeling. CONCLUSION: ARHGAP24 may regulate pseudopod formation downstream of activated ARF6 in MDA-MB-231 human breast carcinoma cells.