The Guanine Nucleotide Exchange Factor Trio Activates the Phagocyte NADPH Oxidase in the Absence of GDP to GTP Exchange on Rac

Sigal, Natalia; Gorzalczany, Yara; Sarfstein, Rive; Weinbaum, Carolyn; Zheng, Yi; Pick, Edgar

doi:10.1074/jbc.m211011200

Cited by 25 publications

(30 citation statements)

References 40 publications

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“…For most of the experiments to follow, complexes were constructed from Rac(1 or 2) exchanged to mant(GDP) before prenylation. These exhibited properties identical to those prepared from prenylated native Rac(1 or 2), which is in the GDP-bound form (73). Under the chromatographic conditions used, Rac1 (pI ϭ 8.77) and Rac2 (pI ϭ 7.52) did not bind to the column.…”

Section: Generation Of Rac1(gdp)⅐rhogdi and Rac2(gdp)⅐rhogdi Complexementioning

confidence: 68%

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Liposomes Comprising Anionic but Not Neutral Phospholipids Cause Dissociation of Rac(1 or 2)·RhoGDI Complexes and Support Amphiphile-independent NADPH Oxidase Activation by Such Complexes

Ugolev

Molshanski-Mor

Weinbaum

et al. 2006

Journal of Biological Chemistry

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Activation of the phagocyte NADPH oxidase involves the assembly of a membrane-localized cytochrome b 559 with the cytosolic components p47 phox , p67 phox , p40 phox , and the GTPase Rac (1 or 2). In resting phagocytes, Rac is found in the cytosol as a prenylated protein in the GDP-bound form, associated with the Rho GDP dissociation inhibitor (RhoGDI). In the process of NADPH oxidase activation, Rac is dissociated from RhoGDI and translocates to the membrane, in concert with the other cytosolic components. The mechanism responsible for dissociation of Rac from RhoGDI is poorly understood. We generated Rac(1 or 2)⅐RhoGDI complexes in vitro from recombinant Rac(1 or 2), prenylated enzymatically, and recombinant RhoGDI, and purified these by anion exchange chromatography. Exposing Rac(1 or 2)(GDP)⅐RhoGDI complexes to liposomes containing four different anionic phospholipids caused the dissociation of Rac(1 or 2)(GDP) from RhoGDI and its binding to the anionic liposomes. Rac2(GDP)⅐RhoGDI complexes were more resistant to dissociation, reflecting the lesser positive charge of Rac2. Liposomes consisting of neutral phospholipid did not cause dissociation of Rac(1 or 2)⅐RhoGDI complexes. Rac1 exchanged to the hydrolysis-resistant GTP analogue, GMPPNP, associated with RhoGDI with lower affinity than Rac1(GDP) and Rac1(GMPPNP)⅐RhoGDI complexes were more readily dissociated by anionic liposomes. Rac1(GMPPNP)⅐RhoGDI complexes elicited NADPH oxidase activation in native phagocyte membrane liposomes in the presence of p67 phox , without the need for an anionic amphiphile, as activator. Both Rac1(GDP)⅐RhoGDI and Rac1(GMPPNP)⅐RhoGDI complexes elicited amphiphileindependent, p67 phox -dependent NADPH oxidase activation in phagocyte membrane liposomes enriched in anionic phospholipids but not in membrane liposomes enriched in neutral phospholipids.

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Section: Generation Of Rac1(gdp)⅐rhogdi and Rac2(gdp)⅐rhogdi Complexementioning

confidence: 68%

“…Identification and Quantification of Nucleotides-Nucleotides bound to native Rac1 or Rac1 exchanged to GMPPNP, present in highly purified Rac1⅐RhoGDI complexes, were released from the protein and identified by high pressure liquid chromatography (HPLC) on a Partisil 10 SAX anion exchange column (Whatman), as described before (73).…”

Section: Methodsmentioning

confidence: 99%

Liposomes Comprising Anionic but Not Neutral Phospholipids Cause Dissociation of Rac(1 or 2)·RhoGDI Complexes and Support Amphiphile-independent NADPH Oxidase Activation by Such Complexes

Ugolev

Molshanski-Mor

Weinbaum

et al. 2006

Journal of Biological Chemistry

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“…Activation of the complex involves the interaction between cytosolic subunits p47 phox and p67 phox , followed by their translocation to the plasma membrane along with COOH-terminal-prenylated Rac1 (28), where they interact with plasma membrane-bound subunits (2). There is an emerging body of evidence demonstrating that mineralocorticoids, like ANG II, may activate NADPH oxidase in various tissues (14,16,20).…”

Section: Discussionmentioning

confidence: 99%

Low-dose spironolactone reduces reactive oxygen species generation and improves insulin-stimulated glucose transport in skeletal muscle in the TG(mRen2)27 rat

Lastra¹,

Whaley‐Connell²,

Manrique³

et al. 2008

American Journal of Physiology-Endocrinology and Metabolism

104

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JR. Low-dose spironolactone reduces reactive oxygen species generation and improves insulin-stimulated glucose transport in skeletal muscle in the TG(mRen2)27 rat. Am J Physiol Endocrinol Metab 295: E110 -E116, 2008. First published April 29, 2008 doi:10.1152/ajpendo.00258.2007.-Renin-angiotensin-aldosterone system (RAAS) activation mediates increases in reactive oxygen species (ROS) and impaired insulin signaling. The transgenic Ren2 rat manifests increased tissue renin-angiotensin system activity, elevated serum aldosterone, hypertension, and insulin resistance. To explore the role of aldosterone in the pathogenesis of insulin resistance, we investigated the impact of in vivo treatment with a mineralocorticoid receptor (MR) antagonist on insulin sensitivity in Ren2 and aged-matched Sprague-Dawley (SD) control rats. Both groups (age 6 -8 wk) were implanted with subcutaneous time-release pellets containing spironolactone (0.24 mg/day) or placebo over 21 days. Systolic blood pressure (SBP) and intraperitoneal glucose tolerance test were determined. Soleus muscle insulin receptor substrate-1 (IRS-1), tyrosine phosphorylated IRS-1, protein kinase B (Akt) phosphorylation, GLUT4 levels, and insulin-stimulated 2-deoxyglucose uptake were evaluated in relation to NADPH subunit expression/oxidase activity and ROS production (chemiluminescence and 4-hydroxy-2-nonenal immunostaining). Along with increased soleus muscle NADPH oxidase activity and ROS, there was systemic insulin resistance and reduced muscle IRS-1 tyrosine phosphorylation, Akt phosphorylation/activation, and GLUT4 expression in the Ren2 group (each P Ͻ 0.05). Despite not decreasing blood pressure, low-dose spironolactone treatment improved soleus muscle insulin signaling parameters and systemic insulin sensitivity in concert with reductions in NADPH oxidase subunit expression/activity and ROS production (each P Ͻ 0.05). Our findings suggest that aldosterone contributes to insulin resistance in the transgenic Ren2, in part, by increasing NADPH oxidase activity in skeletal muscle tissue. Ren2 rat; mineralocorticoid receptor blockade THE ROLES OF INSULIN RESISTANCE in the pathophysiology of type 2 diabetes mellitus (T2DM) and the metabolic syndrome are well established (33,38), and exploration of the mechanisms leading to diminished insulin sensitivity is a field of active research.

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“…Supplementation with Rac1-GTP␥S mutants A27K or G30S or with two small GTPases of the Rho subfamily, CDC42Hs or RhoA (both in GTP␥S-bound form), all of which are unable to interact with p67 phox (29 -31), served as negative controls. As apparent in Table I, only Rac1-GTP␥S and the dominant positive Rac1 mutant Q61L, known to be constitutively in the GTP-bound form (39,44), were able to repair the inactive chimeras.…”

Section: Restoration Of Activity Of Inactive Chimeras Requires a Switmentioning

confidence: 99%

“…38, and identifying the nucleotides by anion exchange chromatography on a Partisil 10 SAX column, as described in Ref. 39.…”

Section: Methodsmentioning

confidence: 99%

Dual Role of Rac in the Assembly of NADPH Oxidase, Tethering to the Membrane and Activation of p67

Sarfstein

Gorzalczany

Mizrahi

et al. 2004

Journal of Biological Chemistry

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124

147

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NADPH oxidase activation involves the assembly of membrane-localized cytochrome b 559 with the cytosolic components p47 phox , p67 phox , and the small GTPase Rac. Assembly is mimicked by a cell-free system consisting of membranes and cytosolic components, activated by an anionic amphiphile. We reported that a chimeric construct, consisting of residues 1-212 of p67 phox and fulllength Rac1, activates the oxidase in vitro in an amphiphile-dependent manner, and when prenylated, in the absence of amphiphile and p47 phox . We subjected chimera p67 phox -(1-212)-Rac1 to mutational analysis and found that: 1) replacement of a single basic residue at the C terminus of the Rac1 moiety by glutamine is sufficient for loss of activity by the non-prenylated chimera; replacement of all six basic residues by glutamines is required for loss of activity by the prenylated chimera. 2) A V204A mutation in the activation domain of the p67 phox moiety leads to a reduction in activity. 3) Mutating residues, known to participate in the interaction between free p67 phox and Rac1, in the p67 phox -(R102E) or Rac1 (A27K, G30S) moieties of the chimera, leads to a marked decrease in activity, indicating a requirement for intrachimeric bonds, in addition to the engineered fusion. 4) Chimeras, inactive because of mutations A27K or G30S in the Rac1 moiety, are reactivated by supplementation with exogenous Rac1-GTP but not with exogenous p67 phox . This demonstrates that Rac has a dual role in the assembly of NADPH oxidase. One is to tether p67 phox to the membrane; the other is to induce an "activating" conformational change in p67 phox . . production, is probably the consequence of a conformational change in gp91 phox , caused by the interaction of cytochrome b 559 with one or several of the cytosolic oxidase components (8). Establishing contact with cytochrome b 559 requires the translocation to the plasma membrane of p47 phox , p67 phox , and Rac, a process known as oxidase assembly (reviewed in Refs. 9 and 10). Stimulus-elicited activation of the oxidase in intact cells can be mimicked in a cell-free system, in which phagocyte membranes or purified and relipidated cytochrome b 559 are mixed with the cytosolic components p47 phox , p67 phox , and Rac, in the presence of an anionic amphiphile, serving as an activator (11-15). The amphiphile acts by causing a conformational change in p47 phox (16) and, possibly, also by the induction of structural changes in cytochrome b 559 (17).The impairment of O 2 . production in the p47 phox -deficient form of chronic granulomatous disease shows that p47 phox is * This work was supported by the Julius Friedrich Cohnheim-Minerva Center for Phagocyte Research, the Ela Kodesz Institute of Host Defense against Infectious Diseases, Israel Science Foundation Grant 428/01, the Roberts-Guthman Chair in Immunopharmacology (to E. P.), and National Institutes of Health Grant GM46372 (to C. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefor...

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The Guanine Nucleotide Exchange Factor Trio Activates the Phagocyte NADPH Oxidase in the Absence of GDP to GTP Exchange on Rac

Cited by 25 publications

References 40 publications

Liposomes Comprising Anionic but Not Neutral Phospholipids Cause Dissociation of Rac(1 or 2)·RhoGDI Complexes and Support Amphiphile-independent NADPH Oxidase Activation by Such Complexes

Liposomes Comprising Anionic but Not Neutral Phospholipids Cause Dissociation of Rac(1 or 2)·RhoGDI Complexes and Support Amphiphile-independent NADPH Oxidase Activation by Such Complexes

Low-dose spironolactone reduces reactive oxygen species generation and improves insulin-stimulated glucose transport in skeletal muscle in the TG(mRen2)27 rat

Dual Role of Rac in the Assembly of NADPH Oxidase, Tethering to the Membrane and Activation of p67

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