Analysis of Protein Phosphorylation in Human Neutrophils

2007 ◽  
pp. 85-96 ◽  
Author(s):  
Jamel El-Benna ◽  
Pham My-Chan Dang
Keyword(s):  
Protein Phosphorylation ◽  
Human Neutrophils

scholarly journals Changes in diacylglycerol labeling, cell shape, and protein phosphorylation distinguish “triggering” from “activation” of human neutrophils.

1988 ◽  
Vol 263 (13) ◽  
pp. 6322-6328 ◽  
Author(s):  
J Reibman ◽  
H M Korchak ◽  
L B Vosshall ◽  
K A Haines ◽  
A M Rich ◽  
...  
Keyword(s):  
Protein Phosphorylation ◽  
Cell Shape ◽  
Human Neutrophils

Surface-reactive stimuli selectively increase protein phosphorylation in human neutrophils

FEBS Letters ◽  
1981 ◽  
Vol 127 (1) ◽  
pp. 4-8 ◽  
Author(s):  
Claudio Schneider ◽  
Margherita Zanetti ◽  
Domenico Romeo
Keyword(s):  
Protein Phosphorylation ◽  
Human Neutrophils

scholarly journals Phorbol ester-induced actin assembly in neutrophils: role of protein kinase C.

1992 ◽  
Vol 116 (3) ◽  
pp. 695-706 ◽  
Author(s):  
G P Downey ◽  
C K Chan ◽  
P Lea ◽  
A Takai ◽  
S Grinstein
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Actin Cytoskeleton ◽  
Protein Phosphorylation ◽  
Neutrophil Activation ◽  
Human Neutrophils ◽  
Actin Assembly ◽  
Membrane Ruffling ◽  
Kinase C

The shape changes and membrane ruffling that accompany neutrophil activation are dependent on the assembly and reorganization of the actin cytoskeleton, the molecular basis of which remains to be clarified. A role of protein kinase C (PKC) has been postulated because neutrophil activation, with the attendant shape and membrane ruffling changes, can be initiated by phorbol esters, known activators of PKC. It has become apparent, however, that multiple isoforms of PKC with differing substrate specificities exist. To reassess the role of PKC in cytoskeletal reorganization, we compared the effects of diacylglycerol analogs and of PKC antagonists on kinase activity and on actin assembly in human neutrophils. Ruffling of the plasma membrane was assessed by scanning EM, and spatial redistribution of filamentous (F)-actin was assessed by scanning confocal microscopy. Staining with NBD-phallacidin and incorporation of actin into the Triton X-100-insoluble ("cytoskeletal") fraction were used to quantify the formation of (F)-actin. [32P]ATP was used to detect protein phosphorylation in electroporated cells. Exposure of neutrophils to 4 beta-PMA (an activator of PKC) induced protein phosphorylation, membrane ruffling, and assembly and reorganization of the actin cytoskeleton, whereas the 4a-isomer, which is inactive towards PKC, failed to produce any of these changes. Moreover, 1,2-dioctanoylglycerol, mezerein, and 3-(N-acetylamino)-5-(N-decyl-N-methylamino)-benzyl alcohol, which are nonphorbol activators of PKC, also promoted actin assembly. Although these effects were consistent with a role of PKC, the following observations suggested that stimulation of conventional isoforms of the kinase were not directly responsible for actin assembly: (a) Okadaic acid, an inhibitor of phosphatases 1 and 2A, potentiated PMA-induced protein phosphorylation, but not actin assembly; and (b) PMA-induced actin assembly and membrane ruffling were not prevented by the conventional PKC inhibitors 1-(5-isoquinolinesulfonyl)-2-methylpiperazine, staurosporine, calphostin C, or sphingosine at concentrations that precluded PMA-induced protein phosphorylation and superoxide production. On the other hand, PMA-induced actin assembly was inhibited by long-chain fatty acid coenzyme A esters, known inhibitors of nuclear PKC (nPKC). We conclude that PMA-induced actin assembly is unlikely to be mediated by the conventional isoforms of PKC, but may be mediated by novel isoforms of the kinase such as nPKC.

scholarly journals Endogenous protein phosphorylation by resting and activated human neutrophils

Blood ◽  
1983 ◽  
Vol 61 (2) ◽  
pp. 333-340 ◽  
Author(s):  
PC Andrews ◽  
BM Babior
Keyword(s):  
Nadph Oxidase ◽  
Protein Phosphorylation ◽  
Time Course ◽  
Normal Subjects ◽  
Protein Band ◽  
Specific Protein ◽  
Striking Similarity ◽  
Human Neutrophils ◽  
Resting Cells ◽  
Opsonized Zymosan

Abstract NADPH oxidase is an enzyme in the plasma membrane of the neutrophil that catalyzes the production of O2-, a species central to the oxygen- dependent killing mechanisms of this cell. The oxidase is dormant in resting cells and is activated upon the addition of a stimulus. Neutrophils of patients with chronic granulomatous disease (CGD) manifest no oxidase activity when stimulated. The possible role of protein phosphorylation in the activation of NADPH oxidase was examined in normal and CGD neutrophils by measuring the incorporation of 32Pi into proteins as determined by gel electrophoresis followed by autoradiography. Resting neutrophils from normal subjects exhibit at least 40 distinct phosphoprotein bands. The level of phosphorylation of these bands was examined after the addition of phorbol myristate acetate (PMA), opsonized zymosan, digitonin, N-formyl-methionyl- phenylalanine (FMLP), or NaF. PMA and opsonized zymosan increased the phosphorylation of a set of 6 protein bands. Digitonin and FMLP consistently caused the phosphorylation of 4 of these protein bands, while NaF failed to induce increased phosphorylation of any protein band. All activators tested caused the dephosphorylation of one specific protein band. The time course of phosphorylation (dephosphorylation) was examined using PMA as the activating agent. Increased phosphorylation of one protein band was evident by 12 sec after the addition of PMA. The most slowly phosphorylated protein band did not slow evidence of change until 5 min after the addition of PMA. Three of the phosphoproteins examined were phosphorylated either earlier than or concomitant with the activation of NADPH oxidase. CGD neutrophils were compared with normal cells for their ability to phosphorylate proteins in response to PMA. The phosphoprotein banding patterns of CGD neutrophils were identical with those of normal neutrophils in both the resting and activated states. The evidence presented shows that the phosphorylation of proteins is a prominent feature of neutrophil metabolism. The striking similarity of phosphorylation changes induced by the various activators tested suggests that protein phosphorylation may play a role in some aspects of neutrophil activation. Evidence was not obtained, however, regarding a link between protein phosphorylation and activation of NADPH oxidase.

scholarly journals Staurosporine inhibits the respiratory burst and induces exocytosis in human neutrophils

1989 ◽  
Vol 264 (3) ◽  
pp. 879-884 ◽  
Author(s):  
B Dewald ◽  
M Thelen ◽  
M P Wymann ◽  
M Baggiolini
Keyword(s):  
Vitamin B12 ◽  
Protein Phosphorylation ◽  
Phorbol Myristate Acetate ◽  
Respiratory Burst ◽  
Cytochalasin B ◽  
Binding Protein ◽  
Human Neutrophils ◽  
Myristate Acetate ◽  
Chemotactic Peptides ◽  
Protein Kinase C Inhibitor

The protein kinase C inhibitor staurosporine influenced in different ways the functions of human neutrophils. Staurosporine prevented the enhanced protein phosphorylation in phorbol ester- and N-formylmethyionyl-leucylphenylalanine (fMLP)-stimulated cells, and was a powerful inhibitor of the respiratory burst induced by phorbol myristate acetate [IC50 (concentration causing 50% inhibition) 17 nM] and the chemotactic peptides fMLP and C5a (IC50 24 nM). It did not alter, however, the superoxide production by cell-free preparations of NADPH oxidase. Staurosporine had no effect on agonist-dependent changes in cytosolic free Ca2+ and exocytosis of specific and azurophil granules, and showed only a slight inhibition of the release of vitamin B12-binding protein induced by phorbol myristate acetate (decreased by 40% at 200 nM). On the other hand, staurosporine also exhibited neutrophil-activating properties: it induced the release of gelatinase (from secretory vesicles) and vitamin-B12-binding protein (from specific granules). These effects were protracted, concentration-dependent, insensitive to Ca2+ depletion, and strongly enhanced by cytochalasin B. Staurosporine, however, did not induce the release of beta-glucuronidase or elastase (from azurophil granules). Except for the sensitivity to cytochalasin B, these properties suggest a similarity between the exocytosis-inducing actions of staurosporine and PMA. The results obtained with staurosporine provide further evidence that different signal-transduction processes are involved in neutrophil activation, and suggest that protein phosphorylation is required for the induction of the respiratory burst, but not for exocytosis.

scholarly journals Endogenous protein phosphorylation by resting and activated human neutrophils

Blood ◽  
1983 ◽  
Vol 61 (2) ◽  
pp. 333-340 ◽  
Author(s):  
PC Andrews ◽  
BM Babior
Keyword(s):  
Nadph Oxidase ◽  
Protein Phosphorylation ◽  
Time Course ◽  
Normal Subjects ◽  
Protein Band ◽  
Specific Protein ◽  
Striking Similarity ◽  
Human Neutrophils ◽  
Resting Cells ◽  
Opsonized Zymosan

NADPH oxidase is an enzyme in the plasma membrane of the neutrophil that catalyzes the production of O2-, a species central to the oxygen- dependent killing mechanisms of this cell. The oxidase is dormant in resting cells and is activated upon the addition of a stimulus. Neutrophils of patients with chronic granulomatous disease (CGD) manifest no oxidase activity when stimulated. The possible role of protein phosphorylation in the activation of NADPH oxidase was examined in normal and CGD neutrophils by measuring the incorporation of 32Pi into proteins as determined by gel electrophoresis followed by autoradiography. Resting neutrophils from normal subjects exhibit at least 40 distinct phosphoprotein bands. The level of phosphorylation of these bands was examined after the addition of phorbol myristate acetate (PMA), opsonized zymosan, digitonin, N-formyl-methionyl- phenylalanine (FMLP), or NaF. PMA and opsonized zymosan increased the phosphorylation of a set of 6 protein bands. Digitonin and FMLP consistently caused the phosphorylation of 4 of these protein bands, while NaF failed to induce increased phosphorylation of any protein band. All activators tested caused the dephosphorylation of one specific protein band. The time course of phosphorylation (dephosphorylation) was examined using PMA as the activating agent. Increased phosphorylation of one protein band was evident by 12 sec after the addition of PMA. The most slowly phosphorylated protein band did not slow evidence of change until 5 min after the addition of PMA. Three of the phosphoproteins examined were phosphorylated either earlier than or concomitant with the activation of NADPH oxidase. CGD neutrophils were compared with normal cells for their ability to phosphorylate proteins in response to PMA. The phosphoprotein banding patterns of CGD neutrophils were identical with those of normal neutrophils in both the resting and activated states. The evidence presented shows that the phosphorylation of proteins is a prominent feature of neutrophil metabolism. The striking similarity of phosphorylation changes induced by the various activators tested suggests that protein phosphorylation may play a role in some aspects of neutrophil activation. Evidence was not obtained, however, regarding a link between protein phosphorylation and activation of NADPH oxidase.

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