Dissociation of Rac1(GDP)·RhoGDI Complexes by the CooperativeAction of Anionic Liposomes Containing Phosphatidylinositol3,4,5-Trisphosphate, Rac Guanine Nucleotide Exchange Factor, andGTP

摘要

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 [] []). 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 GTPanalogue, cause dissociation of Rac1(GDP)·RhoGDI complexes, GDP to GTPexchange on Rac1, and binding of Rac1(GTP) to the liposomes. Complexes werenot dissociated in the absence of GEF and GTP, and optimal dissociationrequired the presence of PtdIns(3,4,5)P3 in the liposomes.Dissociation of Rac1(GDP)·RhoGDI complexes was correlated with theaffinity of particular GEF constructs, via the N-terminal pleckstrin homologydomain, for PtdIns(3,4,5)P3 and involved GEF-mediated GDP to GTPexchange on Rac1. Phagocyte membranes enriched in PtdIns(3,4,5)P3responded by NADPH oxidase activation upon exposure in vitro toRac1(GDP)·RhoGDI complexes, p67^phox, GTP, and RacGEF constructs with affinity for PtdIns(3,4,5)P3 at a levelsuperior to that of native membranes.