p40  Is Phosphorylated on Threonine 154 and Serine 315 during Activation of the Phagocyte NADPH Oxidase

Bouin, Anne-Pascale; Grandvaux, Nathalie; Vignais, Pierre V.; Fuchs, Alexandra

doi:10.1074/jbc.273.46.30097

Cited by 101 publications

(81 citation statements)

References 23 publications

(30 reference statements)

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“…However, we could not determine which part of the PB1 domain participates in the PX-PB1 interaction of p40 phox , because ␤-5 is part of a ␤-sheet core structure and deletion or changes of residues 318-328 to alanines could affect the global folding of the PB1 domain of p40 phox . It is interesting that sites of phosphorylation in p40 phox (Thr154 and Ser315) lie close to the PX domain and residues 318-328 (Bouin et al, 1998). Phosphorylation of Thr154, but not Ser315, was proposed as a negative regulator of ROS production by Nox2 (Lopes et al, 2004).…”

Section: Discussionmentioning

confidence: 99%

“…There are reports that phosphorylation occurs within p40 phox at Thr154 and Ser315 (Bouin et al, 1998) and that phosphorylation of Thr154, but not Ser315, inhibits Nox2 activation (Lopes et al, 2004). Therefore, we made mutants of GFP-p40 phox that could not be phosphorylated [GFPp40 phox (T154A) and GFP-p40 phox (S315A)] or mimic the phosphorylated states [GFP-p40 phox (T154D) and GFP-p40 phox (S315D)].…”

Section: Molecular Biology Of the Cell 444mentioning

confidence: 99%

“…These data suggest that AA induces conformational changes in p40 phox that render the PX domain accessible to bind to early endosomes, as occurs in AA-treated p47 phox to expose its SH3 domains to bind to p22 phox (Shiose and Sumimoto, 2000;Zhao et al, 2002). It is intriguing that GFP-p40 phox and GFP-p47 phox , but not GFP-p67 phox , have the ability to bind to the membrane only after AA stimulation, which likely induces conformational change in both proteins.There are reports that phosphorylation occurs within p40 phox at Thr154 and Ser315 (Bouin et al, 1998) and that phosphorylation of Thr154, but not Ser315, inhibits Nox2 activation (Lopes et al, 2004). Therefore, we made mutants of GFP-p40 phox that could not be phosphorylated [GFPp40 phox (T154A) and GFP-p40 phox (S315A)] or mimic the phosphorylated states [GFP-p40 phox (T154D) and GFP-p40 phox (S315D)].…”

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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

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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...

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Section: Discussionmentioning

confidence: 99%

Section: Molecular Biology Of the Cell 444mentioning

confidence: 99%

mentioning

confidence: 99%

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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

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“…The presence of two phosphorylation sites, Ser-359/ 370, surrounding a polyproline motif crucial for the organization of the ternary complex suggested that phosphorylation may modify the interactions among the cytosolic factors during activation. Several groups suggested a possible switch from one SH3 to the other (24) during the phosphorylation process (17,27,58). At the molecular level, very little is known on the evolution of the interaction network between the cytosolic factors following phosphorylation.…”

Section: P40 Phox Interacts With a Pxxp And A Non-pxxp Motif Of P47mentioning

confidence: 99%

Effects of p47 C Terminus Phosphorylations on Binding Interactions with p40 and p67

Massenet

Chenavas

Cohen‐Addad

et al. 2005

Journal of Biological Chemistry

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The neutrophil NADPH oxidase produces superoxide anions in response to infection. This reaction is activated by association of cytosolic factors, p47 phox and p67 . These data reveal that SH3 p40 can interact with a consensus polyproline motif but also with a noncanonical motif of the p47 phox C terminus. The electrostatic surfaces of both SH3 are very different, and therefore the binding preference for C-SH3 p67 can be attributed to the polyproline motif recognition and particularly to the Arg-368 p47 binding mode. The noncanonical motif contributes equally to interaction with both SH3. The influence of serine phosphorylation on residues 359/ 370 and 379 on the affinity for both SH3 domains has been checked. We conclude that contrarily to previous suggestions, phosphorylation of Ser-359/370 does not modify the SH3 binding affinity for both SH3, whereas phosphorylation of Ser-379 has a destabilizing effect on both interactions. Other mechanisms than a phosphorylation induced switch between the two SH3 must therefore take place for NADPH oxidase activation cascade to start.

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“…The SH3 domain of p40 phox can compete with the C-terminal SH3 domain of p67 phox for binding to the proline rich region of p47 phox [66,67], which suggests some sort of rearrangement of binding partners must occur upon assembly and activation. When cells are activated, p40 phox becomes phosphorylated [68] and translocates along with the p47 phox -p67 phox complex to the membrane [32,33]. The PX domain of p40 phox binds to phosphatidylinositol-3 phosphate and other 3-phosphorylated lipids in the membrane [56,58,69], helping to anchor the other phox regulatory components to the membrane in cells in which PI 3-kinase has been activated [32].…”

Section: Domain Structures Of the Phox Regulatory Subunitsmentioning

confidence: 99%

Regulation of Nox and Duox enzymatic activity and expression

Lambeth

Kawahara

Diebold

2007

Free Radical Biology and Medicine

452

422

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SummaryIn recent years, it has become clear that reactive oxygen species (ROS, which include superoxide, hydrogen peroxide and other metabolites) are produced in biological systems. Rather than being simply a byproduct of aerobic metabolism, it is now recognized that specific enzymes ---the Nox (NADPH-oxidase) and Duox (Dual oxidase) enzymes ----seem to have the sole function of generating ROS in a carefully regulated manner, and key roles in signal transduction, immune function, hormone biosynthesis and other normal biological functions are being uncovered. The prototypical Nox is the respiratory burst oxidase or phagocyte oxidase, which generates large amounts of superoxide and other reactive species in the phagosomes of neutrophils and macrophages, playing a central role in innate immunity by killing microbes. This enzyme system has been extensively studied over the past two decades, and provides a basis for comparison with the more recently described Nox and Duox enzymes, which generate ROS in a variety of cells and tissues. This review first considers the structure and regulation of the respiratory burst oxidase, and then reviews recent studies relating to the regulation of the activity of the novel Nox/Duox enzymes. The regulation of Nox and Duox expression in tissues and by specific stimuli is also considered here. An accompanying review considers biological and pathological roles of the Nox family of enzymes. The Respiratory Burst Oxidase of Phagocytes a. The respiratory burstThe "respiratory burst" refers to the early observation that when professional phagocytes such as neutrophils and macrophages are exposed to microbes, they consume large amounts of oxygen. Unexpectedly, this oxygen consumption was not inhibited by cyanide, an inhibitor of mitochondrial electron transport. This observation led to a more than 25 year search for the enzymatic origin of the respiratory burst and to the eventual discovery and molecular characterization of the phagocytic NADPH-oxidase or "respiratory burst oxidase". The phagocyte oxidase generates superoxide via the one electron-reduction of oxygen by NADPH, with secondary production of hydrogen peroxide, HOCl and other activated forms of oxygen. Together, these reactive oxygen species (ROS) participate in host defense by killing invading microbes. b. gp91 phox , the catalytic moiety of the respiratory burst oxidaseThe phagocytic NADPH-oxidase consists of a membrane-localized glycosylated, catalytic subunit, gp91 phox (which has also come to be known as Nox2, a terminology that will be used in this review), along with a second membrane-associated subunit, p22 phox , both depicted in Fig. 1. Nox2 and p22 phox stabilize one another in a tightly associated heterodimer which is referred to as flavocytochrome b 558 . The C-terminal half of Nox2 forms a domain that is Contact information: 148 Whitehead Biomedical Research Building, Department of Pathology and Laboratory Medicine, 615 Michael Street, Atlanta, GA 30322, U.S. A. Phone: 404-727-5875, Fax: 404-712-2979, e-mail: nox...

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p40 Is Phosphorylated on Threonine 154 and Serine 315 during Activation of the Phagocyte NADPH Oxidase

Cited by 101 publications

References 23 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

Effects of p47 C Terminus Phosphorylations on Binding Interactions with p40 and p67

Regulation of Nox and Duox enzymatic activity and expression

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p40 Is Phosphorylated on Threonine 154 and Serine 315 during Activation of the Phagocyte NADPH Oxidase

Cited by 101 publications

References 23 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

Effects of p47 C Terminus Phosphorylations on Binding Interactions with p40 and p67

Regulation of Nox and Duox enzymatic activity and expression

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Product

Resources

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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