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.
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)P(3)) 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)P(3) 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)P(3) and involved GEF-mediated GDP to GTP exchange on Rac1. Phagocyte membranes enriched in PtdIns(3,4,5)P(3) 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)P(3) at a level superior to that of native membranes.
Ugolev
Yelena
Y
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.
Berdichevsky
Yevgeny
Y
Weinbaum
Carolyn
C
Pick
Edgar
E
D000818:Animals; D001798:Blood Proteins / Q000378:metabolism; D004789:Enzyme Activation; D020730:Guanine Nucleotide Dissociation Inhibitors / Q000378:metabolism*; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006165:Guanylyl Imidodiphosphate / Q000378:metabolism; D006168:Guinea Pigs; D008081:Liposomes / Q000378:metabolism*; D051379:Mice; D046912:Multiprotein Complexes / Q000378:metabolism; D019255:NADPH Oxidases / Q000378:metabolism; D009479:Neuropeptides / Q000378:metabolism*; D010713:Phosphatidylcholines / Q000378:metabolism; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism*; D010718:Phosphatidylserines / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D017434:Protein Structure, Tertiary; D000075684:T-Lymphoma Invasion and Metastasis-inducing Protein 1; D020744:rac GTP-Binding Proteins / Q000378:metabolism*; D020830:rac1 GTP-Binding Protein; D062947:rho-Specific Guanine Nucleotide Dissociation Inhibitors
D001798:Blood Proteins; D020730:Guanine Nucleotide Dissociation Inhibitors; D020662:Guanine Nucleotide Exchange Factors; D008081:Liposomes; D046912:Multiprotein Complexes; D009479:Neuropeptides; D010713:Phosphatidylcholines; D018129:Phosphatidylinositol Phosphates; D010718:Phosphatidylserines; D010750:Phosphoproteins; C484602:Rac1 protein, mouse; D000075684:T-Lymphoma Invasion and Metastasis-inducing Protein 1; C513260:Tiam1 protein, mouse; C490975:Trio protein, mouse; C060974:phosphatidylinositol 3,4,5-triphosphate; C039077:platelet protein P47; D062947:rho-Specific Guanine Nucleotide Dissociation Inhibitors; D006165:Guanylyl Imidodiphosphate; C042511:1,2-dioleoylphosphatidylserine; D006160:Guanosine Triphosphate; D019255:NADPH Oxidases; D017346:Protein Serine-Threonine Kinases; D020744:rac GTP-Binding Proteins; D020830:rac1 GTP-Binding Protein; C017251:1,2-oleoylphosphatidylcholine