scholarly journals Relationship between phosphorylation and translocation to the plasma membrane of p47phox and p67phox and activation of the NADPH oxidase in normal and Ca2+-depleted human neutrophils

1993 ◽  
Vol 290 (1) ◽  
pp. 173-178 ◽  
Author(s):  
S Dusi ◽  
V Della Bianca ◽  
M Grzeskowiak ◽  
F Rossi
Keyword(s):  
Plasma Membrane ◽  
Nadph Oxidase ◽  
Phorbol Esters ◽  
Second Messengers ◽  
Human Neutrophils ◽  
Phosphorylated Proteins ◽  
Kinase C ◽  
Activated State ◽  
Phagocyte Oxidase ◽  
Hydrolysis Of

Stimulation of neutrophils with different agonists activates a latent multicomponent NADPH oxidase that reduces molecular oxygen to superoxide anion. Evidence has accumulated that phosphorylation of p47phox (the 47 kDa cytosolic phagocyte oxidase factor) and translocation of the two cytosolic components p47phox and p67phox are essential steps in the activation of NADPH oxidase in response to phorbol esters. We analysed the relationships between activation of the NADPH oxidase and phosphorylation and translocation of p47phox and p67phox in normal and Ca(2+)-depleted neutrophils stimulated by the receptor-mediated agonists formyl-methionyl-leucyl-phenylalanine and concanavalin A. The results produced the following conclusions: (1) Translocation of p47phox and p67phox is an essential mechanism for activation of the NADPH oxidase. (2) A continuous translocation of p47phox and p67phox is necessary to maintain the NADPH oxidase in an activated state. (3) Only a fraction of p47phox and p67phox translocated to the plasma membrane is functional for the activation of the oxidase. (4) Translocation is independent of protein kinase C, and is linked to transmembrane signalling involving Ca2+ transients and production of lipidic second messengers. However, under some conditions, such as in Ca(2+)-depleted neutrophils, translocation can also occur independently of signalling pathways involving production of second messengers from hydrolysis of phospholipids and Ca2+ transients. (5) Phosphorylation of p47phox and p67phox can be quantitatively dissociated from translocation, as staurosporine markedly inhibits phosphorylation but not translocation. (6) The activity of NADPH oxidase is not correlated with the amounts of the phosphorylated proteins present in the plasma membrane.

Glucose suppresses superoxide generation in normal neutrophils: interference in phospholipase D activation

1993 ◽  
Vol 264 (2) ◽  
pp. C402-C410 ◽  
Author(s):  
J. Ortmeyer ◽  
V. Mohsenin
Keyword(s):  
Nadph Oxidase ◽  
Phosphatidic Acid ◽  
Phospholipase D ◽  
Respiratory Burst ◽  
Second Messengers ◽  
Osmotic Effect ◽  
Acid Generation ◽  
Kinase C ◽  
Fmlp Receptor ◽  
Hydrolysis Of

In neutrophils, N-formyl-Met-Leu-Phe (FMLP) stimulates a respiratory burst with subsequent generation of superoxide anion (O2-.) by NADPH oxidase. Signal transduction involved in this process includes FMLP receptor stimulation of phosphoinositide hydrolysis with formation of inositol 1,4,5-trisphosphate and diacylglycerol and phosphatidylcholine hydrolysis with formation of phosphatidic acid. Generation of these second messengers would lead to activation of NADPH oxidase and generation of O2-.. Neutrophils from diabetic subjects and normal neutrophils exposed to glucose have diminished ability to activate the respiratory burst in response to various agonists. The mechanism of this suppression remains unknown. We report herein that treatment of neutrophils with 15 and 50 mM glucose significantly suppresses the O2-. formation in response to receptor-mediated stimulation. The decreased O2-. generation is associated with marked inhibition of phospholipase D (PLD) activity, with limited hydrolysis of phosphatidylcholine and formation of phosphatidic acid. Sorbitol (50 mM), a nonmetabolizable sugar with a similar osmotic effect, has no influence on O2-. generation or PLD activation. The 4 beta-phorbol 12-myristate 13-acetate (PMA)-induced O2-. generation as well as PLD activation are unaffected by glucose. Furthermore, the intracellular Ca2+ transient in response to FMLP is not influenced by glucose. Taken together, these data suggest that glucose differentially interferes with activation of PLD but not phospholipase C. And, the fact that PMA-induced activation of PLD is not altered by glucose further suggests that a protein kinase C independent step leading to the activation of PLD may be altered by glucose.

scholarly journals Resynthesis of phosphatidylinositol in permeabilized neutrophils following phospholipase Cβ activation: transport of the intermediate, phosphatidic acid, from the plasma membrane to the endoplasmic reticulum for phosphatidylinositol resynthesis is not dependent on soluble lipid carriers or vesicular transport

1999 ◽  
Vol 341 (2) ◽  
pp. 435-444 ◽  
Author(s):  
Jacqueline WHATMORE ◽  
Claudia WIEDEMANN ◽  
Pennti SOMERHARJU ◽  
Philip SWIGART ◽  
Shamshad COCKCROFT
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Vesicular Transport ◽  
Human Neutrophils ◽  
Permeabilized Cells ◽  
Transfer Protein ◽  
Membrane Contact ◽  
Phosphatidylinositol Transfer ◽  
Pi Hydrolysis ◽  
Hydrolysis Of

Receptor-mediated phospholipase C (PLC) hydrolysis of phosphoinositides is accompanied by the resynthesis of phosphatidylinositol (PI). Hydrolysis of phosphoinositides occurs at the plasma membrane, and the resulting diacylglycerol (DG) is converted into phosphatidate (PA). Two enzymes located at the endoplasmic reticulum (ER) function sequentially to convert PA back into PI. We have established an assay whereby the resynthesis of PI could be followed in permeabilized cells. In the presence of [γ-32P]ATP, DG generated by PLC activation accumulates label when converted into PA. The 32P-labelled PA is subsequently converted into labelled PI. The formation of labelled PI reports the arrival of labelled PA from the plasma membrane to the ER. Cytosol-depleted, permeabilized human neutrophils are capable of PI resynthesis following stimulation of PLCβ (in the presence of phosphatidylinositol-transfer protein), provided that CTP and inositol are also present. We also found that wortmannin, an inhibitor of endocytosis, or cooling the cells to 15 °C did not stop PI resynthesis. We conclude that PI resynthesis is dependent neither on vesicular transport mechanisms nor on freely diffusible, soluble transport proteins. Phosphatidylcholine-derived PA generated by the ADP-ribosylation-factor-stimulated phospholipase D pathway was found to accumulate label, reflecting the rapid cycling of PA to DG, and back. This labelled PA was not converted into PI. We conclude that PA derived from the PLC pathway is selected for PI resynthesis, and its transfer to the ER could be membrane-protein-mediated at sites of close membrane contact.

Oncogenes, ions, and phospholipids

1985 ◽  
Vol 248 (1) ◽  
pp. C3-C11 ◽  
Author(s):  
I. G. Macara
Keyword(s):  
Cell Proliferation ◽  
Tyrosine Kinases ◽  
Phorbol Esters ◽  
Second Messengers ◽  
Future Research ◽  
Lipid Biochemistry ◽  
Cytosolic Ph ◽  
Pi Turnover ◽  
Transport Studies ◽  
Kinase C

Recent discoveries in tumor virology, lipid biochemistry, and ion transport studies promise to revolutionize our understanding of cell proliferation, differentiation, and tumorigenesis. A model is proposed, based on similar schemes presented recently by others, that incorporates these discoveries and provides a focus for future research on the functions of oncogene proteins. The model suggests that the early (competence) events in the initiation of cell proliferation are triggered by activation of phosphatidylinositol (PI) turnover, which releases two second messengers, 1,2-diacylglycerol (1,2-DG) and inositol-1,4,5-trisphosphate (IP3). PI turnover is proposed to be regulated by the oncogene protein kinases (src, ros, abl, fps) either directly (acting as PI kinases) or indirectly (as tyrosine kinases). The IP3 triggers Ca2+ release from internal stores, and the elevation of cytosolic Ca2+ acts synergistically with 1,2-DG to activate the Ca2+- and phospholipid-dependent kinase C. Kinase C copurifies with the receptor for the tumor-promoting phorbol esters. It is suggested that kinase C then activates the Na+-H+ exchange system, resulting in an elevation of cytosolic pH and Na+, and that these ionic signals (including the change in Ca2+), either in concert or individually, induce further events, including expression of the protooncogene c-myc, which together commit the cell to initiate replication. Evidences in support of this model are reviewed, together with complications indicating its present inadequacies, particularly recent data suggesting that 1,2-DG may activate tyrosine kinases independent of kinase C.

Lifespan regulation of conventional protein kinase C isotypes

2007 ◽  
Vol 35 (5) ◽  
pp. 1043-1045 ◽  
Author(s):  
D. Carmena ◽  
A. Sardini
Keyword(s):  
Signal Transduction ◽  
Plasma Membrane ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Second Messengers ◽  
Therapeutic Strategies ◽  
Kinase C ◽  
Signalling Network ◽  
Pkc Protein Kinase C ◽  
Lifespan Regulation

Plasma membrane translocation, following allosteric binding of second messengers, initiates the signal transduction process mediated by cPKC [conventional PKC (protein kinase C)] isotypes. Mechanisms regulating the lifespan of the active enzyme such as its phosphorylation, internalization, dephosphorylation and degradation are key elements of the signalling network. The understanding of such mechanisms is essential for the design of therapeutic strategies targeting PKC isoenzymes.

scholarly journals Protein kinase C activity is not involved in N-formylmethionyl-leucyl-phenylalanine-induced phospholipase D activation in human neutrophils, but is essential for concomitant NADPH oxidase activation: studies with a staurosporine analogue with improved selectivity for protein kinase C

1993 ◽  
Vol 292 (3) ◽  
pp. 781-785 ◽  
Author(s):  
G C Kessels ◽  
K H Krause ◽  
A J Verhoeven
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Cyclic Amp ◽  
Nadph Oxidase ◽  
Phospholipase D ◽  
Respiratory Burst ◽  
Feedback Inhibition ◽  
Human Neutrophils ◽  
Kinase C ◽  
Pkc Activation

Stimulation of human neutrophils by the receptor agonist N-formylmethionyl-leucyl-phenylalanine (fMLP) results in a respiratory burst, catalysed by an NADPH oxidase. Concomitantly, phospholipase D (PLD) is activated. To investigate the role of protein kinase C (PKC) in these neutrophil responses, we have compared the effects of staurosporine and a structural analogue of staurosporine (cgp41251), that reflects a higher selectivity towards PKC [Meyer, Regenass, Fabbro, Alteri, Rösel, Müller, Caravatti and Matter (1989) Int. J. Cancer 43, 851-856]. Both staurosporine and cgp41251 dose-dependently inhibited the production of superoxide induced by phorbol 12-myristate 13-acetate (PMA). Both compounds also caused inhibition of the fMLP-induced respiratory burst, but with a lower efficacy during the initiation phase of this response. This latter observation cannot be taken as evidence against PKC involvement in the activation of the respiratory burst, because pretreatment of neutrophils with ionomycin before PMA stimulation also results in a lower efficacy of inhibition. Activation of PLD by fMLP was enhanced in the presence of staurosporine, but not in the presence of cgp41251. Enhancement of PLD activation was also observed in the presence of H-89, an inhibitor of cyclic-AMP-dependent protein kinase (PKA). Both staurosporine and H-89 reversed the dibutyryl-cyclic-AMP-induced inhibition of PLD activation, whereas cgp41251 was without effect. These results indicate that the potentiating effect of staurosporine on PLD activation induced by fMLP does not reflect a feedback inhibition by PKC activation, but instead a feedback inhibition by PKC activation. Taken together, our results indicate that in human neutrophils: (i) PKC activity is not essential for fMLP-induced activation of PLD; (ii) PKC activity does play an essential role in the activation of the respiratory burst by fMLP, other than mediating or modulating PLD activation; (iii) there exists a negative-feedback mechanism on fMLP-induced PLD activation by concomitant activation of PKA.

scholarly journals Phorbol ester and bryostatin differentially regulate the hydrolysis of phosphatidylethanolamine in Ha-ras- and raf-oncogene-transformed NIH 3T3 cells

1991 ◽  
Vol 276 (2) ◽  
pp. 505-509 ◽  
Author(s):  
Z Kiss ◽  
U R Rapp ◽  
G R Pettit ◽  
W B Anderson
Keyword(s):  
Phospholipase D ◽  
Second Messengers ◽  
Transformed Cells ◽  
3T3 Cells ◽  
Intact Cells ◽  
Pkc Inhibitor ◽  
Kinase C ◽  
Nih 3T3 ◽  
Hydrolysis Of ◽  
Nih 3T3 Cells

Previously it was reported that transformation of NIH 3T3 fibroblast by the Ha-ras, v-src, v-fms, and A-raf oncogenes decreased the stimulatory effects of phorbol 12-myristate 13-acetate (PMA; ‘TPA’), an activator of protein kinase C (PKC), on the phosphorylation of an endogenous 80 kDa substrate and on 86Rb uptake [Wolfman, Wingrove, Blackshear & Macara (1987) J. Biol. Chem. 262, 16546-16552], as well as on sphingomyelin synthesis [Kiss, Rapp & Anderson (1988) FEBS Lett. 240, 221-226]. Here, we investigated how transformation affects the PMA-stimulated hydrolysis of phosphatidylethanolamine (PtdEtn), a recently characterized mechanism which may contribute to the generation of the second messengers phosphatidic acid and 1,2-diacylglycerol. The effects of PMA were compared with those of bryostatin, a non-tumour-promoter activator of PKC. Transformation of NIH 3T3 cells with Ha-ras, v-raf, or A-raf enhanced the stimulatory effect of PMA on the phospholipase D-mediated hydrolysis of PtdEtn. On the other hand, the effects of bryostatin on PtdEtn hydrolysis were only slightly increased, if at all, in cells transformed with these oncogenes. In crude membrane preparations isolated from these transformed cells, PMA, but not bryostatin, enhanced the combined stimulatory effects of ATP and the GTP analogue guanosine 5′-[gamma-thio]triphosphate on phospholipase D-mediated PtdEtn hydrolysis. The PKC inhibitor 1-(5-isoquinolinesulphonyl)-2-methylpiperazine inhibited the stimulatory effect of PMA only in intact cells. These results indicate that transformation of cells by certain oncogenes differentially affects phospholipase D-mediated hydrolysis of PtdEtn induced by PMA and bryostatin, suggesting that the action of PMA might involve two different mechanisms.

scholarly journals Protein kinase C-β contributes to NADPH oxidase activation in neutrophils

2000 ◽  
Vol 347 (1) ◽  
pp. 285-289 ◽  
Author(s):  
Lodewijk V. DEKKER ◽  
Michael LEITGES ◽  
Gabriel ALTSCHULER ◽  
Nishil MISTRY ◽  
Aileen MCDERMOTT ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Nadph Oxidase ◽  
Specific Inhibitor ◽  
Human Neutrophils ◽  
Cell Fractionation ◽  
Coated Particles ◽  
Kinase C ◽  
Pkc Β ◽  
Nadph Oxidase Activation

We have analysed the involvement of the β isotype of the protein kinase C (PKC) family in the activation of NADPH oxidase in primary neutrophils. Using immunofluorescence and cell fractionation, PKC-β is shown to be recruited to the plasma membrane upon stimulation with phorbol ester and to the phagosomal membrane upon phagocytosis of IgG-coated particles (Fcγ-receptor stimulus). The time course of recruitment is similar to that of NADPH oxidase activation by these stimuli. The PKC-β specific inhibitor 379196 inhibits the response to PMA as well as to IgG-coated bacteria. Partial inhibition occurs between 10 and 100 nM of inhibitor, the concentration at which PKC-β, but not other PKC isotypes, is targeted. Neutrophils isolated from a mouse that lacks PKC-β also showed an inhibition of NADPH oxidase activation by PMA and IgG-coated particles. The level of inhibition is comparable to that achieved with 379196 in human neutrophils. Thus the PKC-β isotype mediates activation of NADPH oxidase by PMA and by stimulation of Fcγ receptors in neutrophils.

Modulation of neutrophil activation by okadaic acid, a protein phosphatase inhibitor

1992 ◽  
Vol 262 (1) ◽  
pp. C39-C49 ◽  
Author(s):  
D. J. Lu ◽  
A. Takai ◽  
T. L. Leto ◽  
S. Grinstein
Keyword(s):  
Nadph Oxidase ◽  
Protein Kinases ◽  
Phorbol Ester ◽  
Okadaic Acid ◽  
Phorbol Esters ◽  
Specific Inhibitor ◽  
Human Neutrophils ◽  
Respiratory Response ◽  
Dependent Inhibition ◽  
Reduced Rate

We determined the effects of okadaic acid (OA), a specific inhibitor of protein phosphatases 1 (PP1) and 2A (PP2A), on protein phosphorylation and on the activation of the NADPH oxidase in human neutrophils. In otherwise unstimulated cells, OA induced phosphoprotein accumulation, revealing the presence of constitutively active protein kinases. Pulse-chase experiments in electropermeabilized cells confirmed that this effect was due, at least in part, to inhibition of dephosphorylation. OA potentiated phosphoprotein accumulation induced by phorbol esters and by the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP). In phorbol ester-stimulated cells, OA prolonged the respiratory response after inhibition of protein kinase C (PKC) with staurosporine, consistent with a reduced rate of dephosphorylation of active phosphorylated components. Similarly, OA delayed the inactivation of the burst after displacement of FMLP from its receptor by a competitive antagonist. This suggests that the substrates of the protein kinases activated by FMLP are dephosphorylated by PP1 and/or PP2A. That phosphatases control the intensity and duration of the respiratory response is suggested by the finding that OA magnified and prolonged the oxidative burst elicited by FMLP. In contrast, pretreatment with OA produced a time-dependent inhibition of the phorbol ester-induced respiratory burst. Under conditions where inhibition of the phorbol ester response was nearly complete, activation by the chemoattractant peptide not only persisted but was in fact accentuated. These findings provide strong evidence that receptor-mediated stimulation of the NADPH oxidase can occur by pathways not involving PKC.

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