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A W Segal

Publications and source records attributed to A W Segal.

At least 19 recordsLinked to original sources
PubMed1999

Characterization and partial purification of a novel neutrophil membrane-associated kinase capable of phosphorylating the respiratory burst component p47phox.

The phosphorylation of p47phox is widely viewed as an important step in the activation of the neutrophil respiratory burst oxidase system. The exact nature of the kinase(s) responsible remains to be elucidated. We show here that such a kinase was detected on neutrophil membranes activated by either PMA or formyl-methionyl-leucyl-phenylalanine. This enzyme is not intrinsic to the neutrophil membrane and could be eluted with 0.5 M NaCl. The kinase activity was partially purified and was found not to be due to the presence of previously suggested kinases, including protein kinase C isotypes, mitogen-activated protein kinase and protein kinase B. Gel filtration and renaturation in substrate gels suggest a molecular mass of between 45 and 51 kDa. The kinase activity was independent of calcium and lipids but was potently inhibited by staurosporine. Treatment with protein phosphatase 2Ac suggested that the kinase was activated by serine/threonine phosphorylation. Phosphopeptide maps indicated that the kinase phosphorylated p47phox on similar sites to those found in vivo. These results indicate that activation of neutrophils by PMA results in the activation of a membrane-associated kinase that may play a part in the regulation of neutrophil NADPH oxidase through its ability to phosphorylate p47phox.

Binding Sites↗
PubMed1999

The major phosphorylation site of the NADPH oxidase component p67phox is Thr233.

Phosphorylation of p67phox was shown to increase two- to three-fold upon stimulation by PMA, N-formylmethionyl-leucylphenylalanine or serum-opsonized zymosan. Phosphopeptide mapping showed one major tryptic peptide for p67phox immunoprecipitated from resting or stimulated cells. In vitro phosphorylation of p67phox by isolated cytosol or mitogen-activated protein kinase also generated the same phosphopeptide. Results of cyanogen bromide digestion and HPLC-MS suggested that Thr233 was the phosphorylated residue. Mutagenesis of Thr233 to alanine resulted in loss of phosphorylation in vitro. In the present work, Thr233 has been identified as the major phosphorylation site of p67phox, which is situated in a proline-rich domain.

Amino Acid Sequence↗
PubMed1998

Activation of the small GTPase rap1 in human neutrophils.

The small GTPase Rap1 is highly expressed in human neutrophils, but its function is largely unknown. Using the Rap1-binding domain of RalGDS (RalGDS-RBD) as an activation-specific probe for Rap1, we have investigated the regulation of Rap1 activity in primary human neutrophils. We found that a variety of stimuli involved in neutrophil activation, including fMet-Leu-Phe (fMLP), platelet-activating factor (PAF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and IgG-coated particles, induce a rapid and transient Rap1 activation. In addition, we found that Rap1 is normally activated in neutrophils from chronic granulomatous disease patients that lack cytochrome b558 or p47phox and have a defective NADPH oxidase system. From these results we conclude that in neutrophils Rap1 is activated independently of respiratory burst induction. Finally, we found that Rap1 is activated by both the Ca2+ ionophore ionomycin and the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA), indicating that phospholipase C (PLC) activation leading to elevated levels of intracellular free Ca2+ and diacylglycerol (DAG) can mediate Rap1 activation. However, inhibition of PLC and Ca2+ depletion only marginally affected fMLP-induced Rap1 activation, suggesting that additional pathways may control Rap1 activation.

Dinoprostone↗
PubMed1998

Cryptic Rac-binding and p21(Cdc42Hs/Rac)-activated kinase phosphorylation sites of NADPH oxidase component p67(phox).

Rac1 is a member of the Rho family of small molecular mass GTPases that act as molecular switches to control actin-based cell morphology as well as cell growth and differentiation. Rac1 and Rac2 are specifically required for superoxide formation by components of the NADPH oxidase. In binding assays, Rac1 interacts directly with p67(phox), but not with the other oxidase components: cytochrome b, p40(phox), or p47(phox) (Prigmore, E., Ahmed, S., Best, A., Kozma, R. , Manser, E., Segal, A. W., and Lim, L. (1995) J. Biol. Chem. 270, 10717-10722). Here, the Rac1/2 interaction with p67(phox) has been characterized further. Rac1 and Rac2 can bind to p67(phox) amino acid residues 170-199, and the N terminus (amino acids 1-192) of p67(phox) can be used as a specific inhibitor of Rac signaling. Deletion of p67(phox) C-terminal sequences (amino acids 193-526), the C-terminal SH3 domain (amino acids 470-526), or the polyproline-rich motif (amino acids 226-236) stimulates Rac1 binding by approximately 8-fold. p21(Cdc42Hs/Rac)-activated kinase (PAK) phosphorylates p67(phox) amino acid residues adjacent to the Rac1/2-binding site, and this phosphorylation is stimulated by deletion of the C-terminal SH3 domain or the polyproline-rich motif. These data suggest a role for cryptic Rac-binding and PAK phosphorylation sites of p67(phox) in control of the NADPH oxidase.

3T3 Cells↗
PubMed1998

Impairment of mycobacterial immunity in human interleukin-12 receptor deficiency.

In humans, interferon gamma (IFN-gamma) receptor deficiency leads to a predisposition to mycobacterial infections and impairs the formation of mature granulomas. Interleukin-12 (IL-12) receptor deficiency was found in otherwise healthy individuals with mycobacterial infections. Mature granulomas were seen, surrounded by T cells and centered with epithelioid and multinucleated giant cells, yet reduced IFN-gamma concentrations were found to be secreted by activated natural killer and T cells. Thus, IL-12-dependent IFN-gamma secretion in humans seems essential in the control of mycobacterial infections, despite the formation of mature granulomas due to IL-12-independent IFN-gamma secretion.

Animals↗
PubMed1998

Identification of cofilin, coronin, Rac and capZ in actin tails using a Listeria affinity approach.

Actin assembly is involved in cell motility and intracellular movement of Listeria monocytogenes. Induction of Listeria actin tails is mediated by the surface protein ActA. The N-terminal domain of ActA is sufficient for this function. Cell components known to play a role in the actin-based motility of Listeria are VASP (vasodilatator-stimulated phosphoprotein), the multiprotein Arp2/3 complex and cofilin. VASP interacts with the central domain of ActA. Proteins interacting with the N-terminal domain of ActA have not been identified. To identify novel host cell components of ActA-induced actin tails, we used bovine brain extracts and an affinity approach with Listeria as matrix. Several known components of Listeria tails were isolated including VASP, Arp3 and cofilin. Cofilin was identified by peptide sequencing, and cofilin recruitment and Listeria tail length were found to be pH-dependent, in agreement with its recently reported role in enhancing actin filament turnover. In addition, three proteins not previously known to be associated with Listeria tails, coronin, Rac and capZ, were identified in our affinity approach. In infected cells, the localization of the identified proteins was studied by immunofluorescence. Our findings suggest that these latter proteins, which are known to play critical roles in cellular actin rearrangements, may also be involved in the dynamics of Listeria-induced actin assembly.

Actin Depolymerizing Factors↗
PubMed1997

Mammalian actin-related protein 2/3 complex localizes to regions of lamellipodial protrusion and is composed of evolutionarily conserved proteins.

Human neutrophils contain a complex of proteins similar to the actin-related protein 2/3 (Arp2/3) complex of Acanthamoeba. We have obtained peptide sequence information for each member of the putative seven-protein complex previously described for Acanthamoeba and human platelets. From the peptide sequences we have identified cDNA species encoding three novel proteins in this complex. We find that in addition to Arp2 and Arp3, this complex contains a relative of the human (Suppressor of Profilin) SOP2Hs protein and four previously unknown proteins. These proteins localize in the cytoplasm of fibroblasts that lack lamellipodia, but are enriched in lamellipodia on stimulation with serum or platelet-derived growth factor. We propose a conserved and dynamic role for this complex in the organization of the actin cytoskeleton.

3T3 Cells↗
PubMed1997

A modulator of rho family G proteins, rhoGDI, binds these G proteins via an immunoglobulin-like domain and a flexible N-terminal arm.

BACKGROUND: The rho family of small G proteins, including rho, rac and cdc42, are involved in many cellular processes, including cell transformation by ras and the organization of the actin cytoskeleton. Additionally, rac has a role in the regulation of phagocyte NADPH oxidase. Guanine nucleotide dissociation inhibitors (GDIs) of the rhoGDI family bind to these G proteins and regulate their activity by preventing nucleotide dissociation and by controlling their interaction with membranes. RESULTS: We report the structure of rhoGDI, determined by a combination of X-ray crystallography and NMR spectroscopy. NMR spectroscopy and selective proteolysis show that the N-terminal 50-60 residues of rhoGDI are flexible and unstructured in solution. The 2.5 A crystal structure of the folded core of rhoGDI, comprising residues 59-204, shows it to have an immunoglobulin-like fold, with an unprecedented insertion of two short beta strands and a 310 helix. There is an unusual pocket between the beta sheets of the immunoglobulin fold which may bind the C-terminal isoprenyl group of rac. NMR spectroscopy shows that the N-terminal arm is necessary for binding rac, although it remains largely flexible even in the complex. CONCLUSIONS: The rhoGDI structure is notable for the existence of both a structured and a highly flexible domain, both of which appear to be required for the interaction with rac. The immunoglobulin-like fold of the structured domain is unusual for a cytoplasmic protein. The presence of equivalent cleavage sites in rhoGDI and the closely related D4/Ly-GDI (rhoGDI-2) suggest that proteolytic cleavage between the flexible and structured regions of rhoGDI may have a role in the regulation of the activity of members of this family. There is no detectable similarity between the structure of rhoGDI and the recently reported structure of rabGDI, which performs the same function as rhoGDI for the rab family of small G proteins.

Amino Acid Sequence↗
PubMed1997

Analysis of glycosylation sites on gp91phox, the flavocytochrome of the NADPH oxidase, by site-directed mutagenesis and translation in vitro.

Flavocytochrome b558 of the NADPH oxidase which generates superoxide in phagocytic cells, is a alpha1 beta1 heterodimer of gp91phox and p22phox, which together form a membrane-spanning electron-transport chain that transfers electrons from NADPH in the cytosol to oxygen. The C-terminal portion of gp91phox is a member of the ferredoxin-NADP+ reductase family of reductases. Little is known of the organization of the N-terminal section of this molecule, which is associated with the two haem structures. It is N-glycosylated, and site-directed mutagenesis has been used to eliminate the five potential N-linked glycosylation consensus sites. Mutated cDNAs were expressed in vitro. This approach provided evidence for glycosylation of residues Asn131, Asn148 and Asn239, but not of Asn96 and Asn429.

Cytochrome b Group↗
PubMed1997

Immunoelectron microscopy shows a clustered distribution of NADPH oxidase components in the human neutrophil plasma membrane.

The NADPH oxidase that produces superoxide in professional phagocytic cells is a flavocytochrome b electron transport chain in the membrane, a heterodimer of gp91phox and p22phox, that is activated by a number of cytosolic proteins, including p47phox, p67phox, and the small GTP-binding protein p21rac, which translocate to the membrane and attach to the flavocytochrome on activation. The components of this oxidase were localized on the cytoplasmic surface of the plasma membrane of adherent unroofed neutrophils by immunolabeling. Components of the NADPH oxidase and p21rac were found together in punctate clusters occupying 0.03-0.1 microm2 of the cytoplasmic surface of the plasma membrane where the density of labeling of the cytosolic components was increased after stimulation with phorbol myristate acetate.

Antibodies, Monoclonal↗
PubMed1997

Deficiency of p67phox, p47phox or gp91phox in chronic granulomatous disease does not impair leucocyte chemotaxis or motility.

Chronic granulomatous disease (CGD) is a syndrome characterized by failure of the NADPH oxidase of phagocytes that generates superoxide, which is central to the microbicidal process. Cytosolic components of this oxidase system include the proteins p67phox and p47phox, deficiencies of which cause the autosomal recessive form of CGD, whereas the X-linked form of the disease is characterized by a deficiency in the plasma membrane component gp91phox. Components of the oxidase system have been reported to be associated with the cytoskeleton and neutrophils from CGD patients have been reported to have a defective chemotactic response in Boyden chambers. Using a chamber that permits the direct observation of cell behaviour in a linear gradient of a chemoattractant, we have analysed the chemotactic response of neutrophils from a patient lacking p67phox; from another lacking p47phox and from a third lacking gp91phox. The results of measuring the speeds and directions of locomotion of the cells show that their speeds are undiminished relative to cells from healthy control subjects and that their directions of migration are at least as strongly biased in the direction of the gradient as those of the control cells. We conclude that these definitive aspects of the chemotactic response are not abnormal in either the autosomal recessive or the X-linked forms of CGD and that they are therefore not factors in the predisposition to infection that characterizes the syndrome.

Adult↗
PubMed1997

Cytosolic phox proteins interact with and regulate the assembly of coronin in neutrophils.

The NADPH oxidase generates microbicidal superoxide in phagocytes, and when defective it leads to chronic granulomatous disease (CGD). Oxidase specific proteins in the cytosol, p47phox and p67phox, as well as the small GTP binding protein p21rac are important for activation of superoxide production. Because the activity of this oxidase is normally tightly restricted to the phagocytic vacuole, and its temporal and spatial organisation might be regulated by cytoskeletal proteins, we examined the cytosolic phox proteins for interactions with cytoskeletal elements. p67phox copurified with a 57 kDa protein, identified as coronin, an actin binding protein that is important for movement and phagocytosis in Dictyostelium. Binding studies revealed that coronin attaches to the C-terminal half of p40phox, a binding partner of p67phox. The phox proteins and coronin had a similar distribution in the cell, and both accumulated around the phagocytic vacuole. PMA activation of adherent neutrophils resulted in a major rearrangement of these proteins, and of actin, which were lost from the periphery of the cell and condensed around the nucleus. The rearrangement of F-actin and coronin in adherent cells, were absent, or markedly diminished, in cells from patients lacking p47phox or p67phox in which an abnormally large proportion of the coronin was present as part of a large complex. The cytosolic phox proteins might play a regulatory role in the reorganisation of the cytoskeleton accompanying superoxide generation.

Actins↗
PubMed1996

Intramembrane bis-heme motif for transmembrane electron transport conserved in a yeast iron reductase and the human NADPH oxidase.

A plasma membrane iron reductase, required for cellular iron acquisition by Saccharomyces cerevisiae, and the human phagocytic NADPH oxidase, implicated in cellular defense, contain low potential plasma membrane b cytochromes that share elements of structure and function. Four critical histidine residues in the FRE1 protein of the iron reductase were identified by site-directed mutagenesis. Individual mutation of each histidine to alanine eliminated the entire heme spectrum without affecting expression of the apoprotein, documenting the specificity of the requirement for the histidine residues. These critical residues are predicted to coordinate a bis-heme structure between transmembrane domains of the FRE1 protein. The histidine residues are conserved in the related gp91(phox) protein of the NADPH oxidase of human granulocytes, predicting the sites of heme coordination in that protein complex. Similarly spaced histidine residues have also been implicated in heme binding by organelle b cytochromes with little overall sequence similarity to the plasma membrane b cytochromes. This bis-heme motif may play a role in transmembrane electron transport by distinct families of polytopic b cytochromes.

Alleles↗
PubMed1996

Stoichiometry of the subunits of flavocytochrome b558 of the NADPH oxidase of phagocytes.

Flavocytochrome b558, the membrane-spanning component of the NADPH oxidase system of phagocytic cells, is composed of two subunits, p22phox and gp91phox (where phox stands for phagocyte oxidase). The stoichiometry of the subunits has been determined for purified flavocytochrome b556 by: (1) densitometry of Coomassie Blue-stained proteins separated by SDS/PAGE, (2) aromatic absorbance at 280 mm by the subunits after separation by gel filtration under denaturing conditions, (3) crosslinking studies with bis[sulphosuccinimidyl]suberate, where the molecular mass of the cross-linked complex was determined by Western blotting, and (4) radiolabelling of pure flavocytochrome b556 on lysine residues with 125I-labelled Bolton-Hunter reagent (N-succinimidyl-3-(4-hydroxy-5-[125I]iodophenyl)propionate), followed by SDS/PAGE and determination of the radioactivity on each subunit. The ratio of p22phox to gp91phox in the purified flavocytochrome b556 was related back to that in the neutrophil membrane by quantitative Western and dot-blotting to ensure that the stoichiometry was maintained during purification. These measurements showed that the two subunits were present in neutrophil membranes in a molar ratio of 1:1.

Cell Membrane↗
PubMed1996

Interactions between cytosolic components of the NADPH oxidase: p40phox interacts with both p67phox and p47phox.

The NADPH oxidase of neutrophils and other bone-marrow-derived phagocytic cells is a multi-component system consisting of a flavocytochrome b in the plasma membrane and at least four cytosolic proteins. Three of the cytosolic proteins contain src homology 3 (SH3) domains, two each in p47phox and p67phox, and one in p40phox. All three translocate from the cytosol to the flavocytochrome in the membrane upon stimulation of the cells. A small G-protein, p21rac, is also involved in activation of the oxidase. The three cytosolic phox proteins occur as a complex in the cytosol and the strongest interaction appeared to be between p67phox and p40phox. We have investigated the interaction between p40phox and the other two cytosolic phox proteins by in vitro binding assays. An affinity-bead approach was used as well as a biosensor technique (surface plasmon resonance). We observed the strongest attachment between p40phox and p67phox where the binding was between the N-terminal half of p67phox and the C-terminal half of p40phox, and did not appear to involve SH3 domains and proline-rich sequences. p40phox also bound p47phox but more weakly than it did p67phox.

Biosensing Techniques↗
PubMed1996

The FRE1 ferric reductase of Saccharomyces cerevisiae is a cytochrome b similar to that of NADPH oxidase.

Plasma membrane preparations from strains of the yeast Saccharomyces cerevisiae gave a reduced minus oxidized spectrum characteristic of a b-type cytochrome and very similar to the spectrum of flavocytochrome b558 of human neutrophils. The magnitude of the signal correlated with the level of ferric reductase activity and the copy number of the FRE1 gene, indicating that the FRE1 protein is a cytochrome b. Sequence similarities with the flavin binding site of flavocytochrome b558 and other members of the ferredoxin-NADP reductase family, together with increased levels of noncovalently bound FAD and iodonitrotetrazolium violet reductase activity in membranes from a yeast strain overexpressing ferric reductase, suggested that the FRE1 protein may also carry a flavin group. Potentiometric titrations indicated that FRE1, like neutrophil NADPH oxidase, has an unusually low redox potential, in the region of -250 mV, and binds CO.

Amino Acid Sequence↗