Continuous Translocation of Rac2 and the NADPH Oxidase Component p67 during Phagocytosis

Bruggen, Robin van; Anthony, Eloise C.; Fernandez‐Borja, Mar; Roos, Dirk

doi:10.1074/jbc.m309284200

Cited by 58 publications

(63 citation statements)

References 35 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…We reported that p47 phox accumulates transiently on phagocytic cups and mature phagosomes during Fc␥R-mediated phagocytosis (Ueyama et al, 2004), consistent with earlier studies showing p47 phox and p67 phox associate with both nascent and mature phagosomes in neutrophils and neutrophil-like PLB-985 cells (Allen et al, 1999;van Bruggen et al, 2004). The present study shows that GFP-p40 phox also transiently associates on phagosomes, although this targeting is initiated at later stages after phagosome sealing.…”

Section: Discussionsupporting

confidence: 79%

A Regulated Adaptor Function of p40^phox: Distinct p67^phoxMembrane Targeting by p40^phoxand by p47^phox

Ueyama

Tatsuno

Kawasaki

et al. 2007

MBoC

101

View full text Add to dashboard Cite

In the phagocytic cell, NADPH oxidase (Nox2) system, cytoplasmic regulators (p47 phox , p67 phox , p40 phox , and Rac) translocate and associate with the membrane-spanning flavocytochrome b 558 , leading to activation of superoxide production. We examined membrane targeting of phox proteins and explored conformational changes in p40 phox that regulate its translocation to membranes upon stimulation. GFP-p40 phox translocates to early endosomes, whereas GFP-p47 phox translocates to the plasma membrane in response to arachidonic acid. In contrast, GFP-p67 phox does not translocate to membranes when expressed alone, but it is dependent on p40 phox and p47 phox for its translocation to early endosomes or the plasma membrane, respectively. Translocation of GFP-p40 phox or GFP-p47 phox to their respective membrane-targeting sites is abolished by mutations in their phox (PX) domains that disrupt their interactions with their cognate phospholipid ligands. Furthermore, GFP-p67 phox translocation to either membrane is abolished by mutations that disrupt its interaction with p40 phox or p47 phox . Finally, we detected a head-to-tail (PX-Phox and Bem1 [PB1] domain) intramolecular interaction within p40 phox in its resting state by deletion mutagenesis, cell localization, and binding experiments, suggesting that its PX domain is inaccessible to interact with phosphatidylinositol 3-phosphate without cell stimulation. Thus, both p40 phox and p47 phox function as diverse p67 phox "carrier proteins" regulated by the unmasking of membrane-targeting domains in distinct mechanisms. INTRODUCTIONIn phagocytic cells, reactive oxygen species (ROS) are produced by NADPH oxidase (Nox2 system). The enzyme is a multiprotein complex assembled from a membrane-spanning flavocytochrome b 558 (composed of gp91 phox [Nox2] and p22 phox ) and four cytoplasmic components (p47 phox , p67 phox , p40 phox , and Rac) (Leto, 1999;Nauseef, 2004;Quinn and Gauss, 2004). In unstimulated phagocytes, the oxidase is dissociated and inactive: the flavocytochrome b 558 is stored on the membranes of intracellular granules (Jesaitis et al., 1990), Rac is maintained in a GDP-bound cytoplasmic complex dimerized with Rho-guanine nucleotide dissociation inhibitor (GDI) , and the other phox proteins associate in a separate ternary cytoplasmic complex (p47 phox -p67 phox -p40 phox ) in a dephosphorylated state (Bolscher et al., 1989;Rotrosen and Leto, 1990;Kuribayashi et al., 2002;Lapouge et al., 2002). During phagocyte activation, intracellular granules containing flavocytochrome b 558 fuse with phagosomes; p47 phox is phosphorylated, thereby inducing conformational changes in p47 phox that promote the interaction of the cytoplasmic complex with the flavocytochrome b 558 ; and Rac dissociates from Rho-GDI and translocates independently to the membrane after exchange of GDP for GTP (Heyworth et al., 1994;Zhao et al., 2003), resulting in generation of superoxide anion by the transfer of electrons from cytoplasmic NADPH to molecular oxygen.Chronic granulomatous disease...

show abstract

Section: Discussionsupporting

confidence: 79%

A Regulated Adaptor Function of p40^phox: Distinct p67^phoxMembrane Targeting by p40^phoxand by p47^phox

Ueyama

Tatsuno

Kawasaki

et al. 2007

MBoC

101

View full text Add to dashboard Cite

show abstract

“…It has been reported that p47 phox and Rac2 can be translocated without any translocation of p67 phox in lipopolysaccharide-stimulated neutrophils, which gives some credence to this hypothesis (15). Recently, Van Bruggen et al (51) found that the translocation of p67 phox and Rac2 is a cyclic process depending on the integrity of the actin network, in which flavocytochrome b 558 -bound p67 phox and Rac2 are continuously exchanged for free p67 phox and Rac2. The contribution of lipid rafts to the efficient activation of NADPH oxidase has been investigated in neutrophils, and suggests that the assembly of the enzyme complex depends on multiple, cooperative binding and signaling interactions that are greatly facilitated in a raft environment (24,52).…”

Section: Discussionmentioning

confidence: 99%

Interleukin-8-induced Priming of Neutrophil Oxidative Burst Requires Sequential Recruitment of NADPH Oxidase Components into Lipid Rafts

Guichard

Pédruzzi

Dewas

et al. 2005

Journal of Biological Chemistry

103

View full text Add to dashboard Cite

The superoxide-producing phagocyte NADPH oxidase consists of a membrane-bound flavocytochrome b 558 , the cytosol factors p47 phox , p67 phox , p40 phox , and the small GTPase Rac2, which translocate to the membrane to assemble the active complex following neutrophil activation. Interleukin-8 (IL-8) does not activate NADPH oxidase, but potentiates the oxidative burst induced by stimuli such as formyl-methionyl-leucyl-phenylalanine (fMLP) via a priming mechanism. The effect of IL-8 on the components of NADPH oxidase during the priming process has never been investigated in human neutrophils. Here we showed that within 3 min, IL-8 treatment enhanced the Btk-and ERK1/2-dependent phosphorylation of p47 phox , as well as the recruitment of flavocytochrome b 558 , p47 phox , and Rac2 into cholesterol-enriched detergent-resistant microdomains (or lipid rafts). Conversely, IL-8 treatment lasting 15 min failed to recruit flavocytochrome b 558 , p47 phox , or Rac2, but did enhance the Btk-and p38 MAPK-dependent phosphorylation and the translocation of p67 phox into detergent-resistant microdomains. Moreover, methyl-␤-cyclodextrin, which disrupts lipid rafts, inhibited IL-8-induced priming in response to fMLP. Our findings indicate that IL-8-induced priming of the oxidative burst in response to fMLP involves a sequential assembly of the NADPH oxidase components in the lipid rafts of neutrophils.Neutrophils are key components of the early innate response against microbial pathogens. Their activation triggers microbiocidal mechanisms, including the rapid production of reactive oxygen species (ROS) 2 known as the oxidative burst, that play a key role in bacterial killing. However, in an excessive and/or inappropriate response, ROS can induce vascular and tissue lesions. Generation of ROS by neutrophils results from the activation of NADPH oxidase, a multicomponent enzyme system that catalyzes the NADPH-dependent reduction of oxygen to the superoxide anion (O 2 . ), which is the precursor of the other ROS. In resting cells, this multicomponent enzyme system is inactive, and its components are dispersed between the cytosol and the membranes. The flavocytochrome b 558 component, which is composed of two subunits, gp91 phox and p22 phox , is located in the plasma membrane and in specific granules. The other components of the NADPH complex (p47 phox , p67 phox , p40 phox , and small G protein Rac2) are cytosol proteins. The activation of neutrophils by various stimuli, such as bacterial N-formyl peptides (fMLP) and phorbol myristate acetate, triggers the phosphorylation of the p47 phox , p67 phox , and p40 phox cytosolic components and their translocation to the plasma membrane, where they interact with flavocytochrome b 558 (1-4). Concomitantly, Rac2 is dissociated from its inhibitor, RhoGDP dissociation inhibitors, and then interacts with flavocytochrome b 558 to form a binding partner for p67 phox (5). The complete assembly of NADPH oxidase components is crucial for O 2 . production (6, 7).The activation of NADPH oxidase is t...

show abstract

“…We recently showed that p40 phox also undergoes conformational changes by disruption of the intramolecular PX-PB1 domain interaction to enable the ternary cytoplasmic complex to bind to PI(3)P-enriched membranes (11). Finally, Rac translocates to phagosomes (12,13) in a GTP-dependent manner (14,15), resulting in generation of superoxide anion by the transfer of electrons from cytoplasmic NADPH to molecular oxygen.…”

Section: N Phagocytic Cells Reactive Oxygen Species (Ros)mentioning

confidence: 99%

Sequential Binding of Cytosolic Phox Complex to Phagosomes through Regulated Adaptor Proteins: Evaluation Using the Novel Monomeric Kusabira-Green System and Live Imaging of Phagocytosis

Ueyama

Kusakabe

Karasawa

et al. 2008

The Journal of Immunology

View full text Add to dashboard Cite

We engineered a method for detecting intramolecular and intermolecular phox protein interactions in cells by fluorescence microscopy using fusion proteins of complementary fragments of a coral fluorescent reporter protein (monomeric Kusabira-Green). We confirmed the efficacy of the monomeric Kusabira-Green system by showing that the PX and PB1 domains of p40phox interact in intact cells, which we suggested maintains this protein in an inactive closed conformation. Using this system, we also explored intramolecular interactions within p47phox and showed that the PX domain interacts with the autoinhibited tandem Src homology 3 domains maintained in contact with the autoinhibitory region, along with residues 341–360. Furthermore, we demonstrated sequential interactions of p67phox with phagosomes involving adaptor proteins, p47phox and p40phox, during FcγR-mediated phagocytosis. Although p67phox is not targeted to phagosomes by itself, p47phox functions as an adaptor for the ternary complex (p47phox-p67phox-p40phox) in early stages of phagocytosis before phagosome closure, while p40phox functions in later stages after phagosomal closure. Interestingly, a mutated “open” form of p40phox linked p47phox to closed phagosomes and prolonged p47phox and p67phox retention on phagosomes. These results indicate that binding of the ternary complex to phagosomes can be temporally regulated by switching between adaptor proteins that have PX domains with distinct lipid-binding specificities.

show abstract

Continuous Translocation of Rac2 and the NADPH Oxidase Component p67 during Phagocytosis

Cited by 58 publications

References 35 publications

A Regulated Adaptor Function of p40^phox: Distinct p67^phoxMembrane Targeting by p40^phoxand by p47^phox

A Regulated Adaptor Function of p40^phox: Distinct p67^phoxMembrane Targeting by p40^phoxand by p47^phox

Interleukin-8-induced Priming of Neutrophil Oxidative Burst Requires Sequential Recruitment of NADPH Oxidase Components into Lipid Rafts

Sequential Binding of Cytosolic Phox Complex to Phagosomes through Regulated Adaptor Proteins: Evaluation Using the Novel Monomeric Kusabira-Green System and Live Imaging of Phagocytosis

Contact Info

Product

Resources

About

Continuous Translocation of Rac2 and the NADPH Oxidase Component p67 during Phagocytosis

Cited by 58 publications

References 35 publications

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

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

Interleukin-8-induced Priming of Neutrophil Oxidative Burst Requires Sequential Recruitment of NADPH Oxidase Components into Lipid Rafts

Sequential Binding of Cytosolic Phox Complex to Phagosomes through Regulated Adaptor Proteins: Evaluation Using the Novel Monomeric Kusabira-Green System and Live Imaging of Phagocytosis

Contact Info

Product

Resources

About

A Regulated Adaptor Function of p40^phox: Distinct p67^phoxMembrane Targeting by p40^phoxand by p47^phox

A Regulated Adaptor Function of p40^phox: Distinct p67^phoxMembrane Targeting by p40^phoxand by p47^phox