The Rac effector p67phox regulates phagocyte NADPH oxidase by stimulating Vav1 guanine nucleotide exchange activity

The phagocyte NADPH oxidase catalyzes the reduction of molecular oxygen to superoxide and is essential for microbial defense. Electron transport through the oxidase flavocytochrome is activated by the Rac effector p67(phox). Previous studies suggest that Vav1 regulates NADPH oxidase activity elicited by the chemoattractant formyl-Met-Leu-Phe (fMLP). We show that Vav1 associates with p67(phox) and Rac2, but not Rac1, in fMLP-stimulated human neutrophils, correlating with superoxide production. The interaction of p67(phox) with Vav1 is direct and activates nucleotide exchange on Rac, which enhances the interaction between p67(phox) and Vav1. This provides new molecular insights into regulation of the neutrophil NADPH oxidase, suggesting that chemoattractant-stimulated superoxide production can be amplified by a positive feedback loop in which p67(phox) targets Vav1-mediated Rac activation.     

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"

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.     

Activation of G-proteins by receptor-stimulated nucleoside diphosphate kinase in Dictyostelium.

Recently, interest in the enzyme nucleoside diphosphate kinase (EC2.7.4.6) has increased as a result of its possible involvement in cell proliferation and development. Since NDP kinase is one of the major sources of GTP in cells, it has been suggested that the effects of an altered NDP kinase activity on cellular processes might be the result of altered transmembrane signal transduction via guanine nucleotide-binding proteins (G-proteins). In the cellular slime mould Dictyostelium discoideum, extracellular cAMP induces an increase of phospholipase C activity via a surface cAMP receptor and G-proteins. In this paper it is demonstrated that part of the cellular NDP kinase is associated with the membrane and stimulated by cell surface cAMP receptors. The GTP produced by the action of NDP kinase is capable of activating G-proteins as monitored by altered G-protein-receptor interaction and the activation of the effector enzyme phospholipase C. Furthermore, specific monoclonal antibodies inhibit the effect of NDP kinase on G-protein activation. These results suggest that receptor-stimulated NDP kinase contributes to the mediation of hormone action by producing GTP for the activation of GTP-binding proteins.     

Targeting of Rac1 to the phagocyte membrane is sufficient for the induction of NADPH oxidase assembly

The superoxide (O(2))-generating NADPH oxidase complex of phagocytes consists of a membrane-associated flavocytochrome (cytochrome b(559)) and four cytosolic proteins, p47(phox), p67(phox), p40(phox), and the small GTPase Rac (Rac1 or -2). NADPH oxidase activation (O(2) production) is elicited as the consequence of assembly of some or all cytosolic components with cytochrome b(559). This process can be reproduced in an in vitro system consisting of phagocyte membranes, p47(phox), p67(phox), and Rac, activated by an anionic amphiphile. We now show that post-translationally processed (prenylated) Rac1 initiates NADPH oxidase assembly, expressed in O(2) production, in a cell-free system containing phagocyte membrane vesicles and p67(phox), in the absence of an activating amphiphile and of p47(phox). Prenylated Cdc42Hs, a GTPase closely related to Rac, is inactive under the same conditions. Results obtained with phagocyte membrane vesicles can be reproduced fully by replacing these with partially purified cytochrome b(559), incorporated in phosphatidylcholine vesicles. Prenylated, but not nonprenylated, Rac1 binds spontaneously to phagocyte membrane vesicles and also to artificial, protein-free, phosphatidylcholine vesicles, a process counteracted by GDP dissociation inhibitor for Rho. Binding of prenylated Rac1 to membrane vesicles is accompanied by the recruitment of p67(phox) to the same location and the formation of an assembled NADPH oxidase complex, producing O(2) upon the addition of NADPH. Amphiphile and p47(phox)-independent NADPH oxidase activation by prenylated Rac1 is inhibited by Rho GDP dissociation inhibitor and by phosphatidylcholine vesicles, both competing with membrane for prenylated Rac1. We conclude that, in vitro, targeting of Rac to the phagocyte membrane is sufficient for the induction of NADPH oxidase assembly, suggesting that the principal or, possibly, the only role of Rac is to recruit cytosolic p67(phox) to the membrane environment, to be followed by the interaction of p67(phox) with cytochrome b(559).     

Activation of halophilic nucleoside diphosphate kinase by a non-ionic osmolyte,trimethylamine N-oxide

The folding and activity of halophilic enzymes are believed to require the presence of salts at high concentrations. When the inactivated nucleoside diphosphate kinase (NDK) from extremely halophilic archaea was incubated with low salt media, no activity was regained over the course of 8 days. When it was incubated with 2 M NaCl or 3 M KCl, however, it gradually regained activity. To our surprise, trimethylamine N-oxide (TMAO) also was able to induce activation at 4.0 M. The enzyme activity and secondary structure of refolded NDK in 4 M TMAO were comparable with those of the native NDK or the refolded NDK in 3.8 M NaCl. TMAO is not an electrolyte, meaning that the presence of concentrated salts is not an absolute requirement, and that charge shielding or ion binding is not a sole factor for the folding and activation of NDK. Although both NaCl and TMAO are effective in refolding NDK, the mechanism of their actions appears to be different: the effect of protein concentration and pH on refolding is qualitatively different between these two, and at pH 8.0 NDK could be refolded in the presence of 4 M TMAO only when low concentrations of NaCl are included.     

Proteomic analysis of Rac1 signaling regulation by guanine nucleotide exchange factors

The small GTPase Rac1 is implicated in various cellular processes that are essential for normal cell function. Deregulation of Rac1 signaling has also been linked to a number of diseases, including cancer. The diversity of Rac1 functioning in cells is mainly attributed to its ability to bind to a multitude of downstream effectors following activation by Guanine nucleotide Exchange Factors (GEFs). Despite the identification of a large number of Rac1 binding partners, factors influencing downstream specificity are poorly defined, thus hindering the detailed understanding of both Rac1's normal and pathological functions. In a recent study, we demonstrated a role for 2 Rac-specific GEFs, Tiam1 and P-Rex1, in mediating Rac1 anti- versus pro-migratory effects, respectively. Importantly, via conducting a quantitative proteomic screen, we identified distinct changes in the Rac1 interactome following activation by either GEF, indicating that these opposing effects are mediated through GEF modulation of the Rac1 interactome. Here, we present the full list of identified Rac1 interactors together with functional annotation of the differentially regulated Rac1 binding partners. In light of this data, we also provide additional insights into known and novel signaling cascades that might account for the GEF-mediated Rac1-driven cellular effects.     

Guanine nucleotide exchange factors: Activators of the Ras superfamily of proteins

Ras proteins function as critical relay switches that regulate diverse signaling pathways between cell surface receptors and the nucleus. Over the past 2-3 years researchers have identified many components of these pathways that mediate Ras activation and effector function. Among these proteins are several guanine nucleotide exchange factors (GEFs), which are responsible for directly interacting with and activating Ras in response to extracellular stimuli. Analogous GEFs regulate Ras-related proteins that serve other diverse cellular functions. In particular, a growing family of proteins (Dbl homology proteins) has recently been identified, which may function as GEFs for the Rho family of Ras-related proteins. This review summarizes our current knowledge of the structure, biochemistry and biology of Ras and Rho family GEFs. Additionally, we describe mechanisms of GEF activation of Ras in signal transduction and address the potential that deregulated GEFs might contribute to malignant transformation through chronic Ras protein activation.     

Mechanism of NADPH oxidase activation by the Rac/Rho-GDI complex

The low molecular weight GTP binding protein Rac is essential to the activation of the NADPH oxidase complex, involved in pathogen killing during phagocytosis. In resting cells, Rac exists as a heterodimeric complex with Rho GDP dissociation inhibitor (Rho-GDI). Two types of interactions exist between Rac and Rho-GDI: a protein-lipid interaction, implicating the polyisoprene of the GTPase, as well as protein-protein interactions. Using the two-hybrid system, we show that nonprenylated Rac1 interacts very weakly with Rho-GDI, pointing to the predominant role of protein-isoprene interaction in complex formation. In the absence of this strong interaction, we demonstrate that three sites of protein-protein interaction, Arg66(Rac)-Leu67(Rac), His103(Rac), and the C-terminal polybasic region Arg183(Rac)-Lys188(Rac), are involved and cooperate in complex formation. When Rac1 mutants are prenylated by expression in insect cells, they all interact with Rho-GDI. Rho-GDI is able to exert an inhibitory effect on the GDP/GTP exchange reaction except in the complex in which Rac1 has a deletion of the polybasic region (Arg183(Rac)-Lys188(Rac)). This complex is, most likely, held together through protein-lipid interaction only. Although able to function as GTPases, the mutants of Rac1 that failed to interact with Rho-GDI also failed to activate the NADPH oxidase in a cell-free assay after loading with GTP. Mutant Leu119(Rac)Gln could both interact with Rho-GDI and activate the NADPH oxidase. The Rac1/Rho-GDI and Rac1(Leu119Gln)/Rho-GDI complexes, in which the GTPases were bound to GDP, were found to activate the oxidase efficiently. These data suggest that Rho-GDI stabilizes Rac in an active conformation, even in the GDP-bound state, and presents it to its effector, the p67phox component of the NADPH oxidase.     

Mechanism of homocysteine-induced Rac1/NADPH oxidase activation in mesangial cells: role of guanine nucleotide exchange factor Vav2.

We have demonstrated that homocysteine (Hcys) stimulates de novo ceramide synthesis and thereby induces NADPH oxidase activation by increase of Rac GTPase activity in rat mesangial cells (RMCs). However, which isofrom of Rac GTPases is involved in Hcys-induced NADPH oxidase activity and what mechanism mediates Hcys-induced Rac GTPase activation remain unknown. The present study first addressed the role of Rac1 and then determined the contribution of a subfamily of Guanine Nucleotide Exchange Factors (GEFs), Vav, to the action of Hcys on Rac and NADPH oxidase activities in RMCs. By small interfering RNA (siRNA), it was found that Rac1-siRNA attenuated Hcys-induced superoxide (O(2)(-)) production. To explore the mechanism activating Rac by Hcys, GEF-Vav was examined. Vav2 was found to be a predominant isoform among Vav family in RMCs. In Vav2-siRNA transfected RMCs, Hcys-induced Rac activity was blocked, which was accompanied by significant reduction of Hcys-induced O(2)(-). production. This Vav2-siRNA also blocked Rac activation induced by C16-Ceramide (C16-Cer), an intermediate lipid product stimulated by Hcys. Furthermore, we found that Hcys induced Vav2 phosphorylation in a time-dependent manner, which could be induced by C16-Cer and blocked by inhibition of de novo ceramide synthesis. These results suggest that Vav2 importantly contributes to Hcys-induced increase in Rac1 activity and consequent activation of NADPH oxidase in RMCs via ceramide-associated tyrosine phosphorylation.     

Plant mitochondrial nucleoside diphosphate kinase is attached to the membrane through interaction with the adenine nucleotide translocator

This study shows that the plant mitochondrial nucleoside diphosphate kinase (mNDPK) localizes to both the intermembrane space and to the mitochondrial inner membrane. We show that mNDPK is very firmly attached to the membrane. Co-immunoprecipitation experiments identified the adenine nucleotide translocator as an interaction partner. This is the first report showing a direct association between these two proteins, although previous studies have shown metabolic cooperation between them. Possible consequences for mitochondrial energy metabolism are discussed.     

Activation of the leukocyte NADPH oxidase by protein kinase C in a partially recombinant cell-free system.

The leukocyte NADPH oxidase is an enzyme present in phagocytes and B lymphocytes that when activated catalyzes the production of O-2 from oxygen at the expense of NADPH. A correlation between the activation of the oxidase and the phosphorylation of p47(PHOX), a cytosolic oxidase component, is well recognized in whole cells, and direct evidence for a relationship between the phosphorylation of this oxidase component and the activation of the oxidase has been obtained in a number of cell-free systems containing neutrophil membrane and cytosol. Using superoxide dismutase-inhibitable cytochrome c reduction to quantify O-2 production, we now show that p47(PHOX) phosphorylated by protein kinase C activates the NADPH oxidase not only in a cell-free system containing neutrophil membrane and cytosol, but also in a system in which the cytosol is replaced by the recombinant proteins p67(PHOX), Rac2, and phosphorylated p47(PHOX), suggesting that neutrophil plasma membrane plus those three cytosolic proteins are both necessary and sufficient for oxidase activation. In both the cytosol-containing and recombinant cell-free systems, however, activation by SDS yielded greater rates of O-2 production than activation by protein kinase C-phosphorylated p47(PHOX), indicating that a system that employs protein kinase C-phosphorylated p47(PHOX) as the sole activating agent, although more physiological than the SDS-activated system, is nevertheless incomplete.     

Co-regulation of constitutive nitric oxide synthases and NADPH oxidase by the small GTPase Rac

Nitric oxide (NO), generated by NO synthases (NOSs), has multifarious roles in signal transduction. Reactive oxygen species (ROS), generated by ubiquitous NADPH oxidases (NOXs), also participate in cellular signaling. However, the coordination of signals conveyed by NO and ROS is poorly understood. We show that the small GTPase Rac, a component of some NOXs, also interacts with and regulates the constitutively-expressed NOSs. Cellular NO and O(2)(-) production increase or decrease together following activation or inhibition of Rac, and Rac inhibition reveals transduction mechanisms that depend upon NO (vasodilation), ROS (actin polymerization) or both (cytoskeletal organization). Thus, signaling by NO and ROS may be coordinated through a common control element.     

Identification of Rac guanine nucleotide exchange factors promoting Lgl1 phosphorylation in glioblastoma

The protein Lgl1 is a key regulator of cell polarity. We previously showed that Lgl1 is inactivated by hyperphosphorylation in glioblastoma as a consequence of PTEN tumour suppressor loss and aberrant activation of the PI 3-kinase pathway; this contributes to glioblastoma pathogenesis both by promoting invasion and repressing glioblastoma cell differentiation. Lgl1 is phosphorylated by atypical protein kinase C that has been activated by binding to a complex of the scaffolding protein Par6 and active, GTP-bound Rac. The specific Rac guanine nucleotide exchange factors that generate active Rac to promote Lgl1 hyperphosphorylation in glioblastoma are unknown. We used CRISPR/Cas9 to knockout PREX1, a PI 3-kinase pathway-responsive Rac guanine nucleotide exchange factor, in patient-derived glioblastoma cells. Knockout cells had reduced Lgl1 phosphorylation, which was reversed by re-expressing PREX1. They also had reduced motility and an altered phenotype suggestive of partial neuronal differentiation; consistent with this, RNA-seq analyses identified sets of PREX1-regulated genes associated with cell motility and neuronal differentiation. PREX1 knockout in glioblastoma cells from a second patient did not affect Lgl1 phosphorylation. This was due to overexpression of a short isoform of the Rac guanine nucleotide exchange factor TIAM1; knockdown of TIAM1 in these PREX1 knockout cells reduced Lgl1 phosphorylation. These data show that PREX1 links aberrant PI 3-kinase signaling to Lgl1 phosphorylation in glioblastoma, but that TIAM1 is also to fill this role in a subset of patients. This redundancy between PREX1 and TIAM1 is only partial, as motility was impaired in PREX1 knockout cells from both patients.     

Activation of H-Ras in the endoplasmic reticulum by the RasGRF family guanine nucleotide exchange factors

Recent findings indicate that in addition to its location in the peripheral plasma membrane, H-Ras is found in endomembranes like the endoplasmic reticulum and the Golgi complex. In these locations H-Ras is functional and can efficiently engage downstream effectors, but little is known about how its activation is regulated in these environments. Here we show that the RasGRF family exchange factors, both endogenous and ectopically expressed, are present in the endoplasmic reticulum but not in the Golgi complex. With the aid of H-Ras constructs specifically tethered to the plasma membrane, endoplasmic reticulum, and Golgi complex, we demonstrate that RasGRF1 and RasGRF2 can activate plasma membrane and reticular, but not Golgi-associated, H-Ras. We also show that RasGRF DH domain is required for the activation of H-Ras in the endoplasmic reticulum but not in the plasma membrane. Furthermore, we demonstrate that RasGRF mediation favors the activation of reticular H-Ras by lysophosphatidic acid treatment whereas plasma membrane H-Ras is made more responsive to stimulation by ionomycin. Overall, our results provide the initial insights into the regulation of H-Ras activation in the endoplasmic reticulum.     

Signaling function of phagocytic NADPH oxidase: Activation of MAP kinase cascades in phagocytosis

Until recently, the production of reactive oxygen species by NADPH oxidase has been considered only in the context of the oxidative damage to pathogens inside the phagosome. However, homologues of phagocytic NADPH oxidase have been found in almost all cell types, where they produce hydrogen peroxide and thereby regulate the initial intracellular stages of MAP kinase cascades. In the present work, the activation of two MAP kinase cascades, p38 and Erk1/2, during phagocytosis has been studied. It was found that phagocytosis activates both cascades. The activation of Erk1/2 is dependent, and the activation of p38 is not dependent, on the activity of NADPH oxidase. Therefore, the activation of MAP kinases in phagocytes during phagocytosis occurs by a mechanism similar to that operating in nonphagocytic cells, indicating the universality of the function of NADPH oxidases in different cell types.     

Putative functions of nucleoside diphosphate kinase in plants and fungi

The putative functions of NDP (nucleoside diaphosphate) kinases from various organisms focusing to fungi and plants are described. The biochemical reactions catalyzed by NDP kinase are as follows. (i) Phosphotransferring activity from mainly ATP to cognate NDPs generating nucleoside triphosphates (NTPs). (ii) Autophosphorylation activity from ATP and GTP. (iii) Protein kinase (phosphotransferring) activity phosphorylating such as myelin basic protein. NDP kinase could function to provide NTPs as a housekeeping enzyme. However, recent works proved possible functions of the NDP kinases in the processes of signal transduction in various organisms, as described below. By use of the extracts of the mycelia of a filamentous fungus Neurospora crassa blue-light irradiation could increase the phosphorylation of a 15-kDa protein, which was purified and identified to be NDP kinase (NDK-1). By use of the etiolated seedlings of Pisum sativum cv Alaska and Oryza sativa red-light irradiation of intact plants increased the phosphorylation of NDP kinase. However, successive irradiation by red–far-red reversed the reaction, indicating that phytochrome-mediated light signals are transduced to the phosphorylation of NDP kinase. NDP kinase localizing in mitochondria is encoded by nuclear genome and different from those localized in cytoplasm. NDP kinase in mitochondria formed a complex with succinyl CoA synthetase. In Spinicia oleraceae two different NDP kinases were detected in the chloroplast, and in Pisum sativum two forms of NDP kinase originated from single species of mRNA could be detected in the choloroplast. However, the function of NDP kinases in the choloroplast is not yet known. In Neurospora crassa a Pro72His mutation in NDP kinase (ndk-1 ^Pro72His ) deficient in the autophosphorylation and protein kinase activity resulted in lacking the light-induced polarity of perithecia. In wild-type directional light irradiation parallel to the solid medium resulted in the formation of the perithecial beak at the top of perithecia, which was designated as light-induced polarity of perithecia. In wild-type in darkness the beak was formed at random places on perithecia, and in ndk ^Pro72His mutant the perithecial beak was formed at random places even under directional light illumination. The introduction of genomic DNA and cDNA for ndk-1 demonstrated that the wild-type DNAs suppressed the mutant phenotype. With all these results except for the demonstration in Neurospora, most of the phenomena are elusive and should be solved in the molecular levels concerning with NDP kinases.