Stimulation of NADPH oxidase by angiotensin II in human neutrophils is mediated by ERK, p38 MAP-kinase and cytosolic phospholipase A2

The role of cytosolic phospholipase A2 (cPLA2) and its mode of activation by opsonized zymosan (OZ) was studied in human neutrophils in comparison with activation by PMA. The activation of cPLA2 by 1 mg/ml OZ or 50 ng/ml PMA is evidenced by its translocation to the membrane fractions on stimulation. This translocation is consistent with dithiothreitol (DTT)-resistant phospholipase A2 (PLA2) activity detected in the membranes of activated cells. Neutrophils stimulated by either OZ or PMA exhibited an immediate stimulation of extracellular-signal-regulated kinases (ERKs). The inhibition of ERKs, DTT-resistant PLA2 and NADPH oxidase activities by the MAP kinase kinase inhibitor PD-98059 indicates that ERKs mediate the activation of cPLA2 and NADPH oxidase stimulated by either OZ or PMA. The protein kinase C (PKC) inhibitor GF-109203X inhibited epidermal growth factor receptor peptide kinase activity, the release of [3H]arachidonic acid, DTT-resistant PLA2 activity and superoxide generation induced by PMA, but did not inhibit any of these activities induced by OZ. PKC activity was similarly inhibited by GF-109203X in membrane fractions separated from neutrophils stimulated by either PMA or OZ. In the presence of the tyrosine kinase inhibitor genistein, ERKs, PLA2 and NADPH oxidase activities were inhibited in cells stimulated by OZ, whereas they were hardly affected in cells stimulated by PMA. The results suggest that the activation of cPLA2 by PMA or OZ is mediated by ERKs. Whereas PMA stimulates ERKs activity through a PKC-dependent pathway, signal transduction stimulated by OZ involves tyrosine kinase activity leading to activation of ERKs via a PKC-independent pathway.

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Role of cytosolic phospholipase A2 in arachidonic acid release of rat-liver macrophages: regulation by Ca2+ and phosphorylation

Biochemical Journal ◽

10.1042/bj3110189 ◽

1995 ◽

Vol 311 (1) ◽

pp. 189-195 ◽

Cited By ~ 41

Author(s):

P Ambs ◽

M Baccarini ◽

E Fitzke ◽

P Dieter

Keyword(s):

Arachidonic Acid ◽

Phospholipase A2 ◽

Map Kinase ◽

Rat Liver ◽

Kinase Inhibitor ◽

Cytosolic Phospholipase A2 ◽

Ionophore A23187 ◽

Liver Macrophages ◽

Cytosolic Phospholipase ◽

Stimulation Of

In this study we have verified the existence of a cytosolic phospholipase A2 (cPLA2) in rat-liver macrophages. Stimulation of these cells with phorbol 12-myristate 13-acetate (PMA), zymosan and lipopolysaccharide (LPS), but not with the Ca(2+)-ionophore A23187, leads to phosphorylation of cPLA2 and activation of mitogen-activated protein (MAP) kinase, supporting the hypothesis that MAP kinase is involved in cPLA2 phosphorylation. We show furthermore, that the tyrosine kinase inhibitor genistein prevents the LPS- but not the PMA- or zymosan-induced phosphorylation of cPLA2 and activation of MAP kinase, indicating that tyrosine kinases participate in LPS- but not in PMA- and zymosan-induced cPLA2 phosphorylation and MAP kinase activation. Phosphorylation of cPLA2 does not strongly correlate with stimulation of the arachidonic acid (AA) cascade: (1) A23187, a potent stimulator of AA release, fails to induce cPLA2 phosphorylation; (2) withdrawal of extracellular Ca2+, which inhibits PMA-stimulated AA release (Dieter, Schulze-Specking and Decker (1988) Eur. J. Biochem. 177, 61-67), has no effect on PMA-induced phosphorylation of cPLA2; (3) LPS induces cPLA2 phosphorylation within minutes, whereas increased AA release upon treatment with LPS is detectable for the first time after 4 h; and (4) genistein, which prevents LPS-induced cPLA2 phosphorylation, does not inhibit AA release in response to LPS. From these data we suggest that a rise in intracellular Ca2+, but not phosphorylation of cPLA2, is essential for activation of the AA cascade in rat-liver macrophages.

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Effect of lipopolysaccharide on mitogen-activated protein kinases and cytosolic phospholipase A2

Biochemical Journal ◽

10.1042/bj3080815 ◽

1995 ◽

Vol 308 (3) ◽

pp. 815-822 ◽

Cited By ~ 36

Author(s):

S I Fouda ◽

T F P Molski ◽

M S E Ashour ◽

R I Sha′afi

Keyword(s):

Phospholipase A2 ◽

Map Kinase ◽

Map Kinases ◽

Kinase Inhibitor ◽

Cytosolic Phospholipase A2 ◽

Mitogen Activated Protein Kinase ◽

Human Neutrophils ◽

Cytosolic Phospholipase ◽

Low Concentrations ◽

Mitogen Activated Protein

The addition of platelet-activating factor (PAF) to human neutrophils increases phosphorylation on tyrosine residues and stimulates the activity of p42erk2 mitogen-activated protein kinase (MAP kinase). This action is rapid and transient. In contrast, p42erk2, p44erk1 and the p40hera MAP kinase isoforms are all not tyrosine phosphorylated or activated in human neutrophils stimulated with low concentrations of lipopolysaccharide (LPS) in combination with serum. In spite of this, the PAF-induced tyrosine phosphorylation and activation of the p42erk2 MAP kinase are greatly potentiated in cells pretreated with LPS. More interestingly, although low concentrations of LPS do not affect MAP kinase isoforms in these cells, they cause the phosphorylation of cytosolic phospholipase A2 (cPLA2), as evidenced by a decrease in the electrophoretic mobility of the enzyme. In addition, this stimulus-induced upward shift in the mobility of the enzyme is not inhibited by the tyrosine kinase inhibitor, genistein. Furthermore, LPS increases the release of arachidonic acid in control and PAF-stimulated human neutrophils. These observations clearly show that cPLA2 can be phosphorylated and activated by kinases other than the currently known MAP kinases. It is proposed that there are MAP kinase-dependent and -independent mechanisms for the phosphorylation of cPLA2.

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A role for p38 MAP kinase in platelet activation by von Willebrand Factor

Thrombosis and Haemostasis ◽

10.1160/th03-02-0083 ◽

2004 ◽

Vol 91 (01) ◽

pp. 102-110 ◽

Cited By ~ 41

Author(s):

Ilaria Canobbio ◽

Stefania Reineri ◽

Fabiola Sinigaglia ◽

Cesare Balduini ◽

Mauro Torti

Keyword(s):

Arachidonic Acid ◽

Phospholipase A2 ◽

Map Kinase ◽

Platelet Activation ◽

Von Willebrand Factor ◽

P38 Map Kinase ◽

Cyclooxygenase Inhibitors ◽

Von Willebrand ◽

Willebrand Factor ◽

Stimulation Of

SummaryPlatelet activation induced by von Willebrand factor (VWF) binding to the membrane GPIb-IX-V receptor involves multiple signal transduction pathways. Among these, recruitment and activation of the FcγRIIA and stimulation of phospholipase A2 represent independent events equally essential to support a complete platelet response. Phospholipase A2 is activated by calcium and by phosphorylation through MAP kinases. In this work, we found that VWF stimulated the rapid and sustained phosphorylation of p38 MAP kinase (p38MAPK). In vitro kinase assay revealed that VWF-stimulated phosphorylation of p38MAPK was associated with increased kinase activity. Binding of VWF to GPIb-IX-V, but not to integrin αIIbβ3, was required to support phosphorylation of p38MAPK. Neither the blockade of the membrane FcγRIIA by a specific monoclonal antibody or the prevention of thromboxane A2 synthesis by cyclooxygenase inhibitors affected VWF-induced p38MAPK activation. However, phosphorylation of p38MAPK was prevented by the tyrosine kinase Syk inhibitor piceatannol. Treatment of platelets with the p38MAPK inhibitor SB203580 totally prevented VWFstimulated platelet aggregation. Moreover, release of arachidonic acid induced by VWF was strongly impaired by inhibition of p38MAPK. We also found thatVWF induced phosphorylation of cytosolic phospholipase A2, and that this process was prevented by the p38MAPK inhibitor SB203580.These results demonstrate that p38MAPK is a key element in the FcγRIIA-independent pathway for VWF-induced platelet activation, and is involved in the stimulation of phospholipase A2 and arachidonic acid release.

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Inhibition of cAMP-Dependent Protein Kinase (PKA) Activates β2-Adrenergic Receptor (β2-AR) Stimulation of Cytosolic Phospholipase A2 (cPLA2) in Atrial Myocytes

Biophysical Journal ◽

10.1016/j.bpj.2009.12.2702 ◽

2010 ◽

Vol 98 (3) ◽

pp. 496a

Author(s):

Mallikarjuna R. Pabbidi ◽

Xiang Ji ◽

Allen M. Samarel ◽

Stephen L. Lipsius

Keyword(s):

Protein Kinase ◽

Phospholipase A2 ◽

Adrenergic Receptor ◽

Cytosolic Phospholipase A2 ◽

Atrial Myocytes ◽

Dependent Protein Kinase ◽

Cytosolic Phospholipase ◽

Dependent Protein ◽

Β2 Adrenergic Receptor ◽

Stimulation Of

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β2-Adrenergic Receptor (β2-AR) Stimulation of Cytosolic Phospholipase A2 (cPLA2) Is Dependent on PKC and IP3-Mediated Ca2+ Signaling in Atrial Myocytes

Biophysical Journal ◽

10.1016/j.bpj.2009.12.2703 ◽

2010 ◽

Vol 98 (3) ◽

pp. 496a

Author(s):

Mallikarjuna R. Pabbidi ◽

Xiang Ji ◽

Allen M. Samarel ◽

Joshua T. Maxwell ◽

Gregory A. Mignery ◽

...

Keyword(s):

Phospholipase A2 ◽

Adrenergic Receptor ◽

Cytosolic Phospholipase A2 ◽

Atrial Myocytes ◽

Cytosolic Phospholipase ◽

Β2 Adrenergic Receptor ◽

Stimulation Of

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Granulocyte-macrophage colony-stimulating factor (GM-CSF) promotes phosphorylation and an increase in the activity of cytosolic phospholipase A2 in human neutrophils

Biochemical Journal ◽

10.1042/bj3130503 ◽

1996 ◽

Vol 313 (2) ◽

pp. 503-508 ◽

Cited By ~ 34

Author(s):

Nabeel NAHAS ◽

Waltraut H. WATERMAN ◽

Ramadan I. SHA'AFI

Keyword(s):

Phospholipase A2 ◽

Cytosolic Phospholipase A2 ◽

Time Dependent ◽

Human Neutrophils ◽

Colony Stimulating Factor ◽

Gm Csf ◽

Cytosolic Phospholipase ◽

Macrophage Colony Stimulating Factor ◽

Macrophage Colony ◽

Stimulating Factor

Incubation of human neutrophils with 500 pM granulocyte-macrophage colony-stimulating factor (GM-CSF) results in a rapid and time-dependent increase in the phosphorylation of cytosolic phospholipase A2 (cPLA2), which was reflected in a slower electrophoretic mobility of the enzyme. The GM-CSF-induced phosphorylation of cPLA2 was accompanied by a parallel and time-dependent increase in the enzyme activity. Preincubation of neutrophils with the tyrosine kinase inhibitor genistein caused inhibition of the GM-CSF-stimulated phosphorylation and activity of cPLA2. Immunoprecipitation of the enzyme following incubation of neutrophils with [32P]Pi shows that cPLA2 is phosphorylated by GM-CSF. Potato acid phosphatase caused dephosphorylation of the enzyme, indicating that cPLA2 is indeed phosphorylated by GM-CSF. The subcellular distribution of cPLA2 in GM-CSF-stimulated neutrophils revealed that the enzyme resides almost completely in the cytosolic fraction. Addition of Ca2+ to the lysis buffer before homogenization results in the translocation of the phosphorylated and the dephosphorylated forms of the enzyme to the membranes. Translocation of cPLA2 was also achieved after incubation with 0.1 μM N-formylmethionyl-leucyl-phenylalanine (fMLP) after GM-CSF stimulation and when neutrophils were challenged with the Ca2+ ionophore A23187. EDTA and EGTA were unable to solubilize the translocated enzyme from the neutrophil membranes, indicating that cPLA2 is attached to the membranes by strong bonds and not merely due to ionic forces exerted by Ca2+. The inability of GM-CSF to promote arachidonic acid mobilization is probably due to the fact that GM-CSF does not cause an increase in intracellular Ca2+, which is necessary for the translocation of the enzyme to the membranes where its substrate(s) reside.

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Thrombin produces phosphorylation of cytosolic phospholipase A2 by a mitogen-activated protein kinase kinase-independent mechanism in the human astrocytoma cell line 1321N1

Biochemical Journal ◽

10.1042/bj3280263 ◽

1997 ◽

Vol 328 (1) ◽

pp. 263-269 ◽

Cited By ~ 28

Author(s):

Marita HERNÁNDEZ ◽

Yolanda BAYÓN ◽

Mariano SÁNCHEZ CRESPO ◽

María Luisa NIETO

Keyword(s):

Phospholipase A2 ◽

Map Kinase ◽

Cell Line ◽

Cytosolic Phospholipase A2 ◽

Thrombin Receptor ◽

Primary Sequence ◽

Jun Kinase ◽

Cytosolic Phospholipase ◽

Astrocytoma Cell ◽

Mitogen Activated Protein

The release of [3H]arachidonic acid was studied in the 1321N1 astrocytoma cell line upon stimulation with thrombin. The effect of thrombin was antagonized by hirudin only when both compounds were added simultaneously, which suggests activation of thrombin receptor. Evidence that the cytosolic phospholipase A2 (cPLA2) takes part in thrombin-induced arachidonate release was provided by the finding that thrombin induced retardation of the mobility of cPLA2 in SDS/polyacrylamide gels, which is a feature of the activation of cPLA2 by mitogen-activated protein (MAP) kinases. Thrombin induced activation of two members of the MAP kinase family whose consensus primary sequence appears in cPLA2, namely p42-MAP kinase and c-Jun kinase. However, the activation of c-Jun kinase preceded the phosphorylation of cPLA2 more clearly than the activation of p42-MAK kinase did. Both cPLA2 and c-Jun kinase activation were not affected by PD-98059, a specific inhibitor of MAP kinase kinases, which indeed completely blocked p42-MAP kinase shift. Heat shock, a well-known activator of c-Jun kinase, also phosphorylated cPLA2 but not p42-MAP kinase. These data indicate the existence in astrocytoma cells of a signalling pathway triggered by thrombin receptor stimulation that activates a kinase cascade acting on the Pro-Leu-Ser-Pro consensus primary sequence, activates cPLA2, and associates the release of arachidonate with nuclear signalling pathways.

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