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J. Biol. Chem., Vol. 283, Issue 32, 22257-22271, August 8, 2008

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Dissociation of Rac1(GDP)·RhoGDI Complexes by the Cooperative Action of Anionic Liposomes Containing Phosphatidylinositol 3,4,5-Trisphosphate, Rac Guanine Nucleotide Exchange Factor, and GTP^*

Yelena Ugolev, Yevgeny Berdichevsky, Carolyn Weinbaum, and Edgar Pick¹

From the Julius Friedrich Cohnheim-Minerva Center for Phagocyte Research and the Ela Kodesz Institute of Host Defense against Infectious Diseases, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel and the Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710

Rac plays a pivotal role in the assembly of the superoxide-generating NADPH oxidase of phagocytes. In resting cells, Rac is found in the cytosol in complex with Rho GDP dissociation inhibitor (RhoGDI). NADPH oxidase assembly involves dissociation of the Rac·RhoGDI complex and translocation of Rac to the membrane. We reported that liposomes containing high concentrations of monovalent anionic phospholipids cause Rac·RhoGDI complex dissociation ( Ugolev, Y., Molshanski-Mor, S., Weinbaum, C., and Pick, E. (2006) J. Biol. Chem. 281, 19204-19219[Abstract/Free Full Text] ). We now designed an in vitro model mimicking membrane phospholipid remodeling during phagocyte stimulation in vivo. We showed that liposomes of "resting cell membrane" composition (less than 20 mol % monovalent anionic phospholipids), supplemented with 1 mol % of polyvalent anionic phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P₃) in conjunction with constitutively active forms of the guanine nucleotide exchange factors (GEFs) for Rac, Trio, or Tiam1 and a non-hydrolyzable GTP analogue, cause dissociation of Rac1(GDP)·RhoGDI complexes, GDP to GTP exchange on Rac1, and binding of Rac1(GTP) to the liposomes. Complexes were not dissociated in the absence of GEF and GTP, and optimal dissociation required the presence of PtdIns(3,4,5)P₃ in the liposomes. Dissociation of Rac1(GDP)·RhoGDI complexes was correlated with the affinity of particular GEF constructs, via the N-terminal pleckstrin homology domain, for PtdIns(3,4,5)P₃ and involved GEF-mediated GDP to GTP exchange on Rac1. Phagocyte membranes enriched in PtdIns(3,4,5)P₃ responded by NADPH oxidase activation upon exposure in vitro to Rac1(GDP)·RhoGDI complexes, p67^phox, GTP, and Rac GEF constructs with affinity for PtdIns(3,4,5)P₃ at a level superior to that of native membranes.

Received for publication, January 28, 2008 , and in revised form, May 1, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grant GM46372 (to C. W.). This work was also supported by the Julius Friedrich Cohnheim-Minerva Center for Phagocyte Research, the Ela Kodesz Institute of Host Defense against Infectious Diseases, Israel Science Foundation Grant 19/05, and the Roberts-Guthman Chair in Immunopharmacology (to E. P.). 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: Dept. of Human Microbiology, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel. Tel.: 972-3-640-7872; Fax: 972-3-642-9119; E-mail: epick{at}post.tau.ac.il.

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