Mutagenesis of an Arginine- and Lysine-rich Domain in the gp91 Subunit of the Phagocyte NADPH-oxidase Flavocytochromeb 558

Biberstine-Kinkade, Karla J.; Yu, Liping; Dinauer, Mary C.

doi:10.1074/jbc.274.15.10451

Cited by 42 publications

(38 citation statements)

References 55 publications

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“…These findings are consistent with previous studies that showed loss of cytosolic NADPH oxidase components from the phagosomal membrane in X-CGD neutrophils, identifying cytochrome b 558 as an essential factor for correct translocation of p47 phox , p67 phox , and Rac2 (18 -20). Furthermore, in PLB-985 X-CGD cells, the translocation of p47 phox , p67 phox , and Rac2 is known to be disturbed (21). Together with the observation that p67GFP and GFP-Rac2 localize correctly in both PLB-985 and PLB-985 X-CGD cells, this identifies the p67GFP and GFP-Rac2 expressing cell lines as suitable models to study the localization of these proteins in more detail.…”

Section: K562 Cells Already Expressing P22mentioning

confidence: 85%

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

Bruggen

Anthony

Fernandez‐Borja

et al. 2004

Journal of Biological Chemistry

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In this study, the translocation of the NADPH oxidase components p67 phox and Rac2 was studied during phagocytosis in living cells. For this purpose, green fluorescent protein (GFP)-tagged versions of these proteins were expressed in the myeloid cell line PLB-985. First, the correct localization of p67GFP and GFP-Rac2 was shown during phagocytosis of serum-treated zymosan by wild-type PLB-985 cells and PLB-985 X-CGD (chronic granulomatous disease) cells, which lack expression of flavocytochrome b 558 . Subsequently, these constructs were used for fluorescence recovery after photobleaching studies to elucidate the turnover of these proteins on the phagosomal membrane. The turnover of p67GFP and GFP-Rac2 proved to be very high, indicating a continuous exchange of flavocytochrome b 558 -bound p67GFP and GFP-Rac2 for cytosolic, free p67GFP and GFP-Rac2. Furthermore, the importance of an intact actin cytoskeleton for correct localization of these proteins was investigated by disrupting the actin cytoskeleton with cytochalasin B. However, cytochalasin B treatment of PLB-985 cells did not alter the localization of p67GFP and GFP-Rac2 once phagocytosis was initiated. In addition, the continuous exchange of flavocytochrome b 558 -bound p67GFP and GFP-Rac2 for cytosolic p67GFP and GFP-Rac2 was still intact in cytochalasin B-treated cells, indicating that the translocation of these proteins does not depend on a rearrangement of the actin cytoskeleton.

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Section: K562 Cells Already Expressing P22mentioning

confidence: 85%

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

Bruggen

Anthony

Fernandez‐Borja

et al. 2004

Journal of Biological Chemistry

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“…In general, ROS generation by Nox4 and Nox2 was dependent on their distinct B loops. The B loop has been recognized as an important structural element for Nox2 function (5,13,19). Interestingly, the previously identified amino acid residues involved in electron flow from NADPH across the membrane are conserved in Nox2 and Nox4 (arginines 73, 80, 91, and 92; leucine 94; and aspartic acid 95 in Nox2).…”

Section: Discussionmentioning

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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“…Furthermore, both mutagenesis of arginine residues in the Nox2 B-loop and peptides corresponding to the Nox2 B-loop inhibited Nox2 activity and prevented p47 phox / p67 phox translocation to the membrane (32)(33)(34). However, the Nox2 B-loop shares considerable homology with that of Nox4 (see below), suggesting a more general role for this loop in Nox function.…”

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confidence: 99%

Nox4 B-loop Creates an Interface between the Transmembrane and Dehydrogenase Domains

Jackson

Kawahara

Nisimoto

et al. 2010

Journal of Biological Chemistry

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By targeting redox-sensitive amino acids in signaling proteins, the NADPH oxidase (Nox) family of enzymes link reactive oxygen species to physiological processes. We previously analyzed the sequences of 107 Nox enzymes and identified conserved regions that are predicted to have important functions in Nox structure or activation. One such region is the cytosolic B-loop, which in Nox1-4 contains a conserved polybasic region. Previous studies of Nox2 showed that certain basic residues in the B-loop are important for activity and translocation of p47 phox /p67 phox , suggesting this region participates in subunit assembly. However, conservation of this region in Nox4, which does not require p47 phox /p67 phox , suggested an additional role for the B-loop in Nox function. Here, we show by mutation of Nox4 B-loop residues that this region is important for Nox4 activity. Fluorescence polarization detected binding between Nox4 B-loop peptide and dehydrogenase domain (K d ‫؍‬ 58 ؎ 12 nM). This interaction was weakened with Nox4 R96E B-loop corresponding to a mutation that also markedly decreases the activity of holo-Nox4. Truncations of the dehydrogenase domain localize the B-loop-binding site to the N-terminal half of the NADPH-binding subdomain. Similarly, the Nox2 B-loop bound to the Nox2 dehydrogenase domain, and both the Nox2 and Nox4 interactions were dependent on the polybasic region of the B-loop. These data indicate that the B-loop is critical for Nox4 function; we propose that the B-loop, by binding to the dehydrogenase domain, provides the interface between the transmembrane and dehydrogenase domains of Nox enzymes.

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Mutagenesis of an Arginine- and Lysine-rich Domain in the gp91 Subunit of the Phagocyte NADPH-oxidase Flavocytochromeb 558

Cited by 42 publications

References 55 publications

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

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

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

Nox4 B-loop Creates an Interface between the Transmembrane and Dehydrogenase Domains

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