Targeting of Rac1 to the Phagocyte Membrane Is Sufficient for the Induction of NADPH Oxidase Assembly

Abstract

phox -independent NADPH oxidase activation by prenylated Rac1 is inhibited by Rho GDP dissociation inhibitor and by phosphatidylcholine vesicles, both competing with membrane for prenylated Rac1. We conclude that, in vitro, targeting of Rac to the phagocyte membrane is sufficient for the induction of NADPH oxidase assembly, suggesting that the principal or, possibly, the only role of Rac is to recruit cytosolic p67 phox to the membrane environment, to be followed by the interaction of p67 phox with cytochrome b 559 .The production of reactive oxygen species represents the major microbicidal mechanism of professional phagocytes. The primordial oxygen radical is superoxide (O 2 . ), 1 and it is produced by the NADPH-derived one-electron reduction of molecular oxygen, by an enzyme complex known as the NADPH oxidase (referred to here as "oxidase"; reviewed in Refs. 1 and 2 phox , and Rac to a critical concentration of an anionic amphiphile (3, 4).Rac1 or -2 is absolutely required for oxidase assembly in the amphiphile-activated cell-free system (5, 6). It is also clearly involved in O 2. production in intact phagocytes, as shown by the inhibitory effect of Rac antisense oligonucleotides (7) and by selective defects in O 2 . production by neutrophils of Rac2-deficient mice (8) and of a patient with an inhibitory mutation in Rac2 (9). It is the consensus opinion that, in the course of oxidase assembly, Rac is translocated to the membrane (10), although lack of translocation (11) or the lack of relevance of translocation to assembly (12) was also claimed. In the cytosol, Rac is found as a C-terminally prenylated (geranylgeranylated) protein (13), forming a heterodimer with the regulatory protein GDP dissociation inhibitor for Rho (Rho GDI) (5). Dissociation of prenylated Rac from Rho GDI was proposed to be an obligatory step preceding translocation of Rac from the cytosol to the membrane (14 -16). The role of Rac in oxidase assembly was studied extensively in the semirecombinant amphiphile-activated cell-free system (4). Both nonprenylated (5, 17) and prenylated (17-19) Rac are capable of supporting oxidase activation in vitro. It was recently suggested (20) that, whereas membrane association of prenylated Rac (Rac1 or -2) is mediated principally by hydrophobic interaction between the geranylgeranyl tail and membrane lipids, nonprenylated Rac1 binds by electrostatic interaction between a C-terminal polybasic region (21) and negative charges on the membrane. We intended to test the hypothesis that binding of Rac to the membrane is a crucial event in the initiation of oxidase assembly. We found indeed that prenylated, but not nonprenylated, Rac1 initiates oxidase assembly and NADPH-dependent O 2 .production in a cell-free system, containing phagocyte membranes and p67 phox , in the absence of an amphiphilic activator

The Guanine Nucleotide Exchange Factor Trio Activates the Phagocyte NADPH Oxidase in the Absence of GDP to GTP Exchange on Rac

“…Nucleotide Exchange-Prenylated Rac1 was subjected to nucleotide exchange to GTP␥S, as described before (11).…”

“…p67 phox , truncated at residue 212 (p67 phox -(1-212)), was produced in Escherichia coli, as described before (11). Nonprenylated Rac1 and Cdc42Hs were expressed in E. coli, as described earlier (19).…”

“…A characteristic of earlier versions of the amphiphileactivated cell-free system is the use of bacterially expressed recombinant Rac, which did not undergo C-terminal geranylgeranylation (prenylation). We recently described oxidase activation in vitro in a system consisting of phagocyte membranes, p67 phox and prenylated Rac1, in the absence of amphiphile and p47 phox (11). Amphiphile-and p47 phox -independent oxidase activation could also be achieved in mixtures of membrane and a chimeric [p67 phox -Rac1] construct prenylated at the C terminus of the Rac1 segment (12).…”

A Regulated Adaptor Function of p40phox: Distinct p67phoxMembrane Targeting by p40phoxand by p47phox

“…CGD patients who lack p47 phox show impaired translocation of p67 phox to the particulate or membrane fraction, whereas CGD patients who lack p67 phox show normal translocation of p47 phox to the particulate fraction, indicating the adaptor function of p47 phox in recruitment of p67 phox to the membrane Dusi et al, 1996;Allen et al, 1999). However, Nox2 activity can be reconstituted in vitro in the absence of p47 phox , when p67 phox and Rac1 are provided in excess (Freeman and Lambeth, 1996;Koshkin et al, 1996) or when p67 phox is adapted with the membrane-binding sequences from Rac1, although GTP-bound Rac is still required for oxidase activation (Gorzalczany et al, 2000;Alloul et al, 2001), indicating p67 phox and Rac1 are minimum essential cytoplasmic components in the Nox2 system. p40 phox also has a PX domain that specifically binds to phosphatidylinositol 3-phosphate [PI(3)P] (Bravo et al, 2001;Kanai et al, 2001), a phospholipid enriched in the early endosome (Gillooly et al, 2000) and produced in the phagosomal membrane during phagocytosis (Ellson et al, 2001a;Gillooly et al, 2001).…”

Inhibition of hypoxia/reoxygenation-induced oxidative stress in HGF-stimulated antiapoptotic signaling: role of PI3-K and Akt kinase upon rac1

“…7 Rac1 belongs to the rho family of small GTP binding proteins and its role in the production of ROS in phagocytic cells such as neutrophils is well established. [9][10][11] In such cells, rac proteins are essential for the assembly of the plasma membrane NADPH oxidase, which is responsible for the transfer of electrons to molecular oxygen leading to the production of superoxide anions. Rac proteins, in particular rac1, function similarly in nonphagocytic cells, 1,12 and such rac1-regulated ROS have been implicated in a variety of cellular processes including growth, migration, and transformation.…”