Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.6.3.1 (NADPH oxidase)
11,281 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The Rho family GTPase Rac1 mediates a variety of signal transduction processes leading to activation of NADPH oxidase, actin cytoskeleton reorganization, transcription activation, and stimulation of DNA synthesis. In this study, Rac1 was found to form a reversible monomer and oligomer in both the GDP- and GTP-bound states in vitro and in cells. Mutational analysis and peptide competition experiments showed that the unique C-terminal domain of Rac1 consisting of six consecutive basic residues (amino acids 183-188) is required for the homophilic interaction. Oligomerization of Rac1-GTP led to a self-stimulatory GTPase-activating protein (GAP) activity, resulting in a significantly enhanced intrinsic GTP hydrolysis rate of Rac1-GTP. Deletion or mutation of the polybasic residues drastically decreased its intrinsic GTPase activity and resulted in a loss of the self-stimulatory GAP activity. In the oligomeric state, Rac1 became insensitive to the RhoGAP stimulation, albeit maintaining the responsiveness to the guanine nucleotide exchange factor. The ability of the Rac1 C-terminal mutants to activate the effector p21(cdc42/rac)-activated kinase-1 correlated with their oligomerization states, suggesting that oligomer formation potentiates effector activation. Furthermore, the oligomer-to-monomer transition of Rac1-GDP could be driven effectively by interaction with the Rho guanine nucleotide dissociation inhibitor. Building on previous characterizations of Rac1 interaction with regulatory proteins and effectors, these results suggest that Rac1 may employ yet another means of regulation by cycling between the monomeric and oligomeric states to effectively generate a transient and augmented signal.
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PMID:Oligomerization of Rac1 gtpase mediated by the carboxyl-terminal polybasic domain. 1113 22

Transient expression of constitutively active Rac1 derivatives, (G12V) or (Q61L), was sufficient to induce phagocyte NADPH oxidase activity in a COS-7 cell model in which human cDNAs for essential oxidase components, gp91(phox), p22(phox), p47(phox), and p67(phox), were expressed as stable transgenes. Expression of constitutively active Rac1 in "COS(phox)" cells induced translocation of p47(phox) and p67(phox) to the membrane. Furthermore, translocation of p47(phox) was induced in the absence of p67(phox) expression, even though Rac does not directly bind p47(phox). Rac effector domain point substitutions (A27K, G30S, D38A, Y40C), which can selectively eliminate interaction with different effector proteins, impaired Rac1V12-induced superoxide production. Activation of endogenous Rac1 by expression of constitutively active Rac-guanine nucleotide exchange factor (GEF) derivatives was sufficient to induce high level NADPH oxidase activity in COS(phox) cells. The constitutively active form of the hematopoietic-specific GEF, Vav1, was the most effective at activating superoxide production, despite detection of higher levels of Rac1-GTP upon expression of constitutively active Vav2 or Tiam1 derivatives. These data suggest that Rac can play a dual role in NADPH oxidase activation, both by directly participating in the oxidase complex and by activating signaling events leading to oxidase assembly, and that Vav1 may be the physiologically relevant GEF responsible for activating this Rac-regulated complex.
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PMID:Rac activation induces NADPH oxidase activity in transgenic COSphox cells, and the level of superoxide production is exchange factor-dependent. 1189 53

The superoxide-generating NADPH oxidase complex of phagocytes consists of a membrane-associated flavocytochrome b(559) and four cytosolic components as follows: p47(phox), p67(phox), p40(phox), and the small GTPase Rac (1 or 2). Activation of the oxidase is the result of assembly of the cytosolic components with cytochrome b(559) and can be mimicked in vitro by mixtures of membrane and cytosolic components exposed to an anionic amphiphile, serving as activator. We reported that prenylation of Rac1 endows it with the ability to support oxidase activation in conjunction with p67(phox) but in the absence of amphiphile and p47(phox). We now show the following 6 points. 1) The Rac guanine nucleotide exchange factor Trio markedly potentiates oxidase activation by prenylated Rac1-GDP. 2) This occurs in the absence of exogenous GTP or any other source of GTP generation, demonstrating that the effect of Trio does not involve GDP to GTP exchange on Rac1. 3) Trio does not potentiate oxidase activation by prenylated Rac1-GTP, by nonprenylated Rac1-GDP in the presence or absence of amphiphile, and by a prenylated [p67(phox)-Rac1] chimera in GDP-bound form. 4) Rac1 mutants defective in the ability to bind Trio or to respond to Trio by nucleotide exchange fail to respond to Trio by enhanced oxidase activation. 5) A Trio mutant with conserved Rac1-binding ability but lacking nucleotide exchange activity fails to enhance oxidase activation. 6) The effect of Trio is mimicked by displacement of Mg(2+) from Rac1-GDP. These results reveal the existence of a novel mechanism of Rac activation by a guanine nucleotide exchange factor and suggest that the induction by Trio of a conformational change in Rac1, in the absence of nucleotide exchange, is sufficient for enhancing its effector function.
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PMID:The guanine nucleotide exchange factor trio activates the phagocyte NADPH oxidase in the absence of GDP to GTP exchange on Rac. "The emperor's nw clothes". 1247 76

Rac plays a central role in regulating neutrophil responses to inflammatory signals, including actin remodeling, chemotaxis, and superoxide production by the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Rac-GTP is a component of the membrane-assembled NADPH oxidase complex, and new evidence suggests that Rac-GTP interacts directly with the oxidase flavocytochrome, in addition to binding to the regulatory p67 subunit, to regulate electron transfer both independently and cooperatively from NADPH to molecular oxygen. Other new studies suggest that Rac-GTP plays a dual role in NADPH oxidase activation, and can initiate signaling pathways leading to translocation of cytosolic oxidase subunits in addition to functioning in the assembled enzyme complex. Rac activation in response to neutrophil chemoattractants may be regulated in large part by a newly identified guanine nucleotide exchange factor, P-Rex1, which is activated by either phosphatidylinositols or Gbetagamma subunits. Multiple Rac GTPase activating proteins are present in neutrophils and may also modulate levels of Rac-GTP. The importance of Rac in a broad range of neutrophil functions is shown by the variety of defects seen in neutrophils from Rac2 knockout mice and from a patient with recurrent infections and a dominant-negative mutation in Rac2.
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PMID:Regulation of neutrophil function by Rac GTPases. 1248 6

Substantial evidence suggests that the transient production of H(2)O(2) is an important signaling event triggered by the activation of various cell surface receptors. Understanding the intracellular messenger function of H(2)O(2) calls for studies of how receptor occupation elicits the production of H(2)O(2), what kinds of molecules are targeted by the produced H(2)O(2), and how H(2)O(2) is eliminated after the completion of its mission. Recent studies suggest that growth factor-induced H(2)O(2) production requires the activation of PtdIns 3-kinase. The essential role of PtdIns 3-kinase is likely to provide PI(3,4,5)P(3) that recruits and activates a guanine nucleotide exchange factor of Rac, which is required for the activation of NADPH oxidase. The targets of H(2)O(2) action include proteins that contain a reactive Cys residue. Thus, H(2)O(2) produced in response to growth factor causes inactivation of protein tyrosine phosphatases in various cells by oxidizing specifically the catalytic Cys. These results, together with other observations, indicate that the activation of a receptor tyrosine kinase per se by binding of the corresponding growth factor might not be sufficient to increase the steady-state level of protein tyrosine phosphorylation in cells. Rather, the concurrent inhibition of protein tyrosine phosphatases by H(2)O(2) might also be required. Peroxiredoxins, members of a newly discovered family of peroxidases, efficiently reduced the intracellular level of H(2)O(2) produced in the cells stimulated with various cell surface ligands. Furthermore, the activity of peroxiredoxin enzymes seems to be regulated via protein phosphorylation as in the case of many other intracellular messenger metabolizing enzymes.
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PMID:Cellular regulation by hydrogen peroxide. 1287 33

Rho GTPases integrate the intracellular signaling in a wide range of cellular processes. Activation of these G proteins is tightly controlled by a number of guanine nucleotide exchange factors (GEFs). In this study, we addressed the functional role of the recently identified p114RhoGEF in in vivo experiments. Activation of endogenous G protein-coupled receptors with lysophosphatidic acid resulted in activation of a transcription factor, serum response element (SRE), that was enhanced by p114RhoGEF. This stimulation was inhibited by the functional scavenger of Gbetagamma subunits, transducin. We have determined that Gbetagamma subunits but not Galpha subunits of heterotrimeric G proteins stimulated p114RhoGEF-dependent SRE activity. Using coimmunoprecipitation assay, we have determined that Gbetagamma subunits interacted with full-length and DH/PH domain of p114RhoGEF. Similarly, Gbetagamma subunits stimulated SRE activity induced by full-length and DH/PH domain of p114RhoGEF. Using in vivo pull-down assays and dominant-negative mutants of Rho GTPases, we have determined that p114RhoGEF activated RhoA and Rac1 but not Cdc42 proteins. Functional significance of RhoA activation was established by the ability of p114RhoGEF to induce actin stress fibers and cell rounding. Functional significance of Rac1 activation was established by the ability of p114RhoGEF to induce production of reactive oxygen species (ROS) followed by activation of NADPH oxidase enzyme complex. In summary, our data showed that the novel guanine nucleotide exchange factor p114RhoGEF regulates the activity of RhoA and Rac1, and that Gbetagamma subunits of heterotrimeric G proteins are activators of p114RhoGEF under physiological conditions. The findings help to explain the integrated effects of LPA and other G-protein receptor-coupled agonists on actin stress fiber formation, cell shape change, and ROS production.
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PMID:G Protein betagamma subunits stimulate p114RhoGEF, a guanine nucleotide exchange factor for RhoA and Rac1: regulation of cell shape and reactive oxygen species production. 1459 93

Human neutrophil adherence to ECMs induces an initial inhibition of stimulated reactive oxygen species (ROS) formation, followed by an enhanced phase of oxidant production. The initial integrin-mediated suppression of ROS constitutes a mechanism to prevent inappropriate tissue damage as leukocytes migrate to inflammatory sites. The Rac2 guanosine 5'-triphosphatase (GTPase) is a critical regulatory component of the phagocyte NADPH oxidase. We show that activation of Rac2 is inhibited in adherent neutrophils, correlating with inhibition of ROS formation. Conversely, NADPH oxidase components p47 and p67 assemble normally, suggesting a specific action of adhesion on the Rac2 molecular switch. Reconstitution with activated Rac2 restored rapid NADPH oxidase activation kinetics to adherent neutrophils, establishing that inhibition was due to defective Rac2 activity. We provide evidence that integrins inhibit Rac2 activation via a membrane-associated guanine nucleotide exchange factor, likely to be Vav1. Activation of Vav1, but not its upstream activator, Syk, is suppressed by cell adhesion. Vav1 activity is inhibited due to dephosphorylation of the regulatory Tyr174 via enhanced tyrosine phosphatase activity in adherent cells. These studies identify an integrin-mediated pathway in which Vav1 is as a strong candidate for the critical regulatory point in suppression of Rac2 activation and ROS generation during inflammatory responses.
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PMID:The molecular basis for adhesion-mediated suppression of reactive oxygen species generation by human neutrophils. 1466 Jul 49

The generation of reactive oxygen species (ROS) in cells stimulated with growth factors requires the activation of phosphatidylinositol 3-kinase (PI3K) and the Rac protein. We report here that the COOH-terminal region of Nox1, a protein related to gp91(phox) (Nox2) of phagocytic cells, is constitutively associated with beta Pix, a guanine nucleotide exchange factor for Rac. Both growth factor-induced ROS production and Rac1 activation were completely blocked in cells depleted of beta Pix by RNA interference. Rac1 was also shown to bind to the COOH-terminal region of Nox1 in a growth factor-dependent manner. Moreover, the depletion of Nox1 by RNA interference inhibited growth factor-induced ROS generation. These results suggest that ROS production in growth factor-stimulated cells is mediated by the sequential activation of PI3K, beta Pix, and Rac1, which then binds to Nox1 to stimulate its NADPH oxidase activity.
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PMID:Sequential activation of phosphatidylinositol 3-kinase, beta Pix, Rac1, and Nox1 in growth factor-induced production of H2O2. 1512 57

Activation of the phagocyte NADPH oxidase is the consequence of the assembly of membranal cytochrome b559 with the cytosolic components p47phox, p67phox, and the GTPase Rac and is mimicked by a cell-free system comprising these components and an activator. We designed a variant of this system, consisting of membranes, p67phox) prenylated Rac1-GDP, and the Rac-specific guanine nucleotide exchange factor (GEF) Trio, in which oxidase activation is induced in the absence of an activator and p47phox. We now show that: 1) Trio and another Rac GEF (Tiam1) act by inducing GDP to GTP exchange on prenylated Rac1-GDP and that our earlier assertion that activation is GTP-independent is explained by contamination of p67phox preparations with GTP and/or ATP. 2) Oxidase activation by Rac GEFs is supported not only by GTP but also by ATP. 3) Non-hydrolysable GTP analogs are active, whereas ATP analogs, incapable of gamma-phosphoryl transfer, are inactive. 4) The ability of ATP to support GEF-induced oxidase activation is explained by ATP serving as a gamma-phosphoryl donor for a membrane-localized nucleoside diphosphate kinase (NDPK), converting GDP to GTP. 5) The existence of a NDPK in macrophage membranes is proven by functional, enzymatic, and immunologic criteria. 6) NDPK acts on free GDP, and the newly formed GTP is bound again to Rac. 7) Free GDP is derived exclusively by dissociation from prenylated Rac1-GDP, mediated by GEF. NDPK and GEF appear to be functionally linked in the sense that the availability of GDP, serving as substrate for NDPK, is dependent on the level of activity of GEF.
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PMID:Activation of the phagocyte NADPH oxidase by Rac Guanine nucleotide exchange factors in conjunction with ATP and nucleoside diphosphate kinase. 1555 78

It is well established that growth-factor-induced reactive oxygen species (ROS) act as second messengers in cell signaling. We have previously reported that betaPix, a guanine nucleotide exchange factor for Rac, interacts with NADPH oxidase 1 (Nox1) leading to EGF-induced ROS generation. Here, we report the identification of the domains of Nox1 and betaPix responsible for the interaction between the two proteins. GST pull-down assays show that the PH domain of betaPix binds to the FAD-binding region of Nox1. We also show that overexpression of the PH domain of betaPix results in inhibition of superoxide anion generation in response to EGF. Additionally, NADPH oxidase Organizer 1 (NoxO1) is shown to interact with the NADPH-binding region of Nox1. These results suggest that the formation of the complex consisting of Nox1, betaPix, and NoxO1 is likely to be a critical step in EGF-induced ROS generation.
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PMID:Molecular interaction of NADPH oxidase 1 with betaPix and Nox Organizer 1. 1632 88


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