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J. Biol. Chem., Vol. 283, Issue 28, 19836-19844, July 11, 2008

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The Pleckstrin Homology Domain of the Arf6-specific Exchange Factor EFA6 Localizes to the Plasma Membrane by Interacting with Phosphatidylinositol 4,5-Bisphosphate and F-actin^*

Eric Macia, Mariagrazia Partisani, Cyril Favard, Eva Mortier^¶, Pascale Zimmermann^¶, Marie-France Carlier^||, Pierre Gounon^**, Frédéric Luton, and Michel Franco¹

From the Institut de Pharmacologie Moléculaire et Cellulaire, UMR 6097 CNRS-Université de Nice-Sophia Antipolis, 660, route des lucioles, 06560 Valbonne, France, Institut Fresnel, CNRS UMR 6133, avenue Escadrille Normandie 13397 Marseille cedex 20, France, ^¶Department Human Genetics, University of Leuven, B-3000 Leuven, Belgium, ^||Laboratoire d'Enzymologie et Biologie Structurales UPR 3082 CNRS-Université, 1 avenue de la Terrasse, 91198 Gif-sur-Yvette, France, and ^**Centre Commun de Microscopie Appliquée, Université de Nice-Sophia Antipolis Parc Valrose, 06103 Nice cedex 2, France

The Arf6-specific exchange factor EFA6 coordinates membrane trafficking with actin cytoskeleton remodeling. It localizes to the plasma membrane where it catalyzes Arf6 activation and induces the formation of actin-based membrane ruffles. We have shown previously that the pleckstrin homology (PH) domain of EFA6 was responsible for its membrane localization. In this study we looked for the partners of the PH domain at the plasma membrane. Mutations of the conserved basic residues suspected to be involved in the binding to phosphoinositides redistribute EFA6-PH to the cytosol. In addition, phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) breakdown also leads to the solubilization of EFA6-PH. Direct binding measured by surface plasmon resonance gives an apparent affinity of 0.5 µM EFA6-PH for PI(4,5)P₂. Moreover, we observed in vitro that the catalytic activity of EFA6 is strongly increased by PI(4,5)P₂. These results indicate that the plasma membrane localization of EFA6-PH is based on its interaction with PI(4,5)P₂, and this interaction is necessary for an optimal catalytic activity of EFA6. Furthermore, we demonstrated by fluorescence recovery after photobleaching and Triton X-100 detergent solubility experiments that in addition to the phophoinositides, EFA6-PH is linked to the actin cytoskeleton. We observed both in vivo and in vitro that EFA6-PH interacts directly with F-actin. Finally, we demonstrated that EFA6 could bind simultaneously filamentous actin and phospholipids vesicles. Our results explain how the exchange factor EFA6 via its PH domain could coordinate at the plasma membrane actin cytoskeleton organization with membrane trafficking.

Received for publication, January 30, 2008 , and in revised form, May 13, 2008.

^* This work was supported by grants from Association pour la Recherche contre le Cancer, Cancéropôle PACA (Provence-Alpes-Côte d'Azur), and Agence Nationale de la Recherche. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed. Tel.: 33-4-93-95-77-70; Fax: 33-4-93-95-77-10; E-mail: franco{at}ipmc.cnrs.fr.

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