Identification of a Conserved Rac-binding Site on NADPH Oxidases Supports a Direct GTPase Regulatory Mechanism

Kao, Yu-Ya; Gianni, Davide; Bohl, Benjamin P.; Taylor, R. M.; Bokoch, Gary M.

doi:10.1074/jbc.m801010200

Cited by 69 publications

(58 citation statements)

References 54 publications

(63 reference statements)

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“…The C-terminal cytoplasmic domain of Nox2 contains binding sites for the oxidase components p47 phox , p67 phox , and the GTPase Rac, which are essential for assembly and activation of a functional Nox2-based oxidase (12,17,18). When exogenously expressed, Nox4 activity is not dependent on these proteins (21).…”

Section: Resultsmentioning

confidence: 99%

Structural Insights into Nox4 and Nox2: Motifs Involved in Function and Cellular Localization

Löhneysen¹,

Noack²,

Wood

et al. 2010

Molecular and Cellular Biology

112

114

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Regulated generation of reactive oxygen species (ROS) is primarily accomplished by NADPH oxidases (Nox).Nox1 to Nox4 form a membrane-associated heterodimer with p22 phox , creating the docking site for assembly of the activated oxidase. Signaling specificity is achieved by interaction with a complex network of cytosolic components. Nox4, an oxidase linked to cardiovascular disease, carcinogenesis, and pulmonary fibrosis, deviates from this model by displaying constitutive H 2 O 2 production without requiring known regulators. Extensive Nox4/Nox2 chimera screening was initiated to pinpoint structural motifs essential for ROS generation and Nox subcellular localization. In summary, a matching B loop was crucial for catalytic activity of both Nox enzymes. Substitution of the carboxyl terminus was sufficient for converting Nox4 into a phorbol myristate acetate (PMA)-inducible phenotype, while Nox2-based chimeras never gained constitutive activity. Changing the Nox2 but not the Nox4 amino terminus abolished ROS generation. The unique heterodimerization of a functional Nox4/p22 phox Y121H complex was dependent on the D loop. Nox4, Nox2, and functional Nox chimeras translocated to the plasma membrane. Cell surface localization of Nox4 or PMA-inducible Nox4 did not correlate with O 2 ؊ generation. In contrast, Nox4 released H 2 O 2 and promoted cell migration. Our work provides insights into Nox structure, regulation, and ROS output that will aid inhibitor design.

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Section: Resultsmentioning

confidence: 99%

Structural Insights into Nox4 and Nox2: Motifs Involved in Function and Cellular Localization

Löhneysen¹,

Noack²,

Wood

et al. 2010

Molecular and Cellular Biology

112

114

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“…phox (35)(36)(37)(38)(39). To test the role of the p67 phox region of amino acids 190 -210 in binding to the C-terminal NADPH-binding domain of gp91 phox (amino acids 384 -570), we performed a GST pull-down assay using purified p67 phox proteins (Fig.…”

Section: Role Of the Region Of Amino Acids 190 -205 In P67mentioning

confidence: 99%

A Conserved Region between the TPR and Activation Domains of p67 Participates in Activation of the Phagocyte NADPH Oxidase

Maehara

Miyano

Yuzawa

et al. 2010

Journal of Biological Chemistry

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The phagocyte NADPH oxidase, dormant in resting cells, is activated during phagocytosis to produce superoxide, a precursor of microbicidal oxidants. The membrane-integrated protein gp91 phox serves as the catalytic core, because it contains a complete electron-transporting apparatus from NADPH to molecular oxygen for superoxide production. Activation of gp91 phox requires the cytosolic proteins p67 phox , p47 phox , and Rac (a small GTPase). p67 phox , comprising 526 amino acids, moves upon cell stimulation to the membrane together with p47 phox and there interacts with Rac; these processes are prerequisite for gp91 phox .

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“…In the view championed in this study, the third stage is represented predominantly by the interaction of p67 phox with Nox2, with p47 phox and Rac, seen as accessory molecules, acting as carriers of p67 phox to the membrane vicinity of Nox2 or as stabilizers of the Nox2-p67 phox bond. The main alternative to this is the proposal of a direct involvement of Rac in the induction of electron flow in Nox2, based on the evidence for binding of Rac2 to Nox2, involving the insert region of Rac (residues 124 -135) (17)(18)(19). Contradictory data on the role of the insert region of Rac in oxidase activation left this issue unsettled (16,20,21).…”

mentioning

confidence: 99%

A Prenylated p47 -p67 -Rac1 Chimera Is a Quintessential NADPH Oxidase Activator

Mizrahi

Berdichevsky

Casey

et al. 2010

Journal of Biological Chemistry

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The superoxide-generating NADPH oxidase complex of resting phagocytes includes cytochrome b 559 , a membrane-associated heterodimer composed of two subunits (Nox2 and p22 phox ), and four cytosolic proteins (p47 phox , p67 phox , Rac, and p40 phox ). Upon stimulation, the cytosolic components translocate to the membrane, as the result of a series of interactions among the cytosolic components and among the cytosolic components and cytochrome b 559 and its phospholipid environment. We described the construction of a tripartite chimera (trimera) consisting of strategic domains of p47 phox , p67 phox , and Rac1, in which interactions among cytosolic components were replaced by fusion (Berdichevsky, Y., Mizrahi, A., Ugolev, Y., Molshanski-Mor, S., and Pick, E. (2007) J. Biol. Chem. 282, 22122-22139). We now fused green fluorescent protein (GFP) to the N terminus of the trimera and found the following. 1) The GFP-p47 phox -p67 phox -Rac1 trimera activates the oxidase in amphiphile-dependent and -independent (anionic phospholipid-enriched membrane) cell-free systems. 2) Geranylgeranylation of the GFP-trimera makes it a potent oxidase activator in unmodified (native) membranes and in the absence of amphiphile. 3) Prenylated GFP-trimera binds spontaneously to native membranes (as assessed by gel filtration and in-line fluorometry), forming a tight complex capable of NADPH-dependent, activator-independent superoxide production at rates similar to those measured in canonical cell-free systems. 4) Prenylation of the GFP-trimera supersedes completely the dependence of oxidase activation on the p47 phox phox homology domain and, partially, on the Rac1 polybasic domain, but the requirement for Trp 193 in p47 phox persists. Prenylated GFP-p47 phox -p67 phoxRac1 trimera acts as a quintessential single molecule oxidase activator of potential use in high throughput screening of inhibitors.

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Identification of a Conserved Rac-binding Site on NADPH Oxidases Supports a Direct GTPase Regulatory Mechanism

Cited by 69 publications

References 54 publications

Structural Insights into Nox4 and Nox2: Motifs Involved in Function and Cellular Localization

Structural Insights into Nox4 and Nox2: Motifs Involved in Function and Cellular Localization

A Conserved Region between the TPR and Activation Domains of p67 Participates in Activation of the Phagocyte NADPH Oxidase

A Prenylated p47 -p67 -Rac1 Chimera Is a Quintessential NADPH Oxidase Activator

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