Phosphorylation of Cytosolic Group IV Phospholipase A2 Is Necessary but Not Sufficient for Arachidonic Acid Release in P388D1 Macrophages

2000 ◽  
Vol 267 (1) ◽  
pp. 145-148 ◽  
Author(s):  
María A. Balboa ◽  
Jesús Balsinde ◽  
Edward A. Dennis
Keyword(s):  
Arachidonic Acid ◽  
Phospholipase A2 ◽  
Group Iv ◽  
Arachidonic Acid Release ◽  
Acid Release

scholarly journals Role of Phosphorylation Sites and the C2 Domain in Regulation of Cytosolic Phospholipase A2

1999 ◽  
Vol 145 (6) ◽  
pp. 1219-1232 ◽  
Author(s):  
Miguel A. Gijón ◽  
Diane M. Spencer ◽  
Alan L. Kaiser ◽  
Christina C. Leslie
Keyword(s):  
Arachidonic Acid ◽  
Nuclear Envelope ◽  
Phospholipase A2 ◽  
Okadaic Acid ◽  
Cytosolic Phospholipase A2 ◽  
Phosphorylation Sites ◽  
C2 Domain ◽  
Arachidonic Acid Release ◽  
Cytosolic Phospholipase ◽  
Acid Release

Cytosolic phospholipase A2 (cPLA2) mediates agonist-induced arachidonic acid release, the first step in eicosanoid production. cPLA2 is regulated by phosphorylation and by calcium, which binds to a C2 domain and induces its translocation to membrane. The functional roles of phosphorylation sites and the C2 domain of cPLA2 were investigated. In Sf9 insect cells expressing cPLA2, okadaic acid, and the calcium-mobilizing agonists A23187 and CryIC toxin induce arachidonic acid release and translocation of green fluorescent protein (GFP)-cPLA2 to the nuclear envelope. cPLA2 is phosphorylated on multiple sites in Sf9 cells; however, only S505 phosphorylation partially contributes to cPLA2 activation. Although okadaic acid does not increase calcium, mutating the calcium-binding residues D43 and D93 prevents arachidonic acid release and translocation of cPLA2, demonstrating the requirement for a functional C2 domain. However, the D93N mutant is fully functional with A23187, whereas the D43N mutant is nearly inactive. The C2 domain of cPLA2 linked to GFP translocates to the nuclear envelope with calcium-mobilizing agonists but not with okadaic acid. Consequently, the C2 domain is necessary and sufficient for translocation of cPLA2 to the nuclear envelope when calcium is increased; however, it is required but not sufficient with okadaic acid.

Evidence for a Role for Phospholipase C, but not Phospholipase A2, in Platelet Activation in Response to Low Concentrations of Collagen

2001 ◽  
Vol 85 (05) ◽  
pp. 882-889 ◽  
Author(s):  
Leslie Lockhart ◽  
Caroline Pampolina ◽  
Brent Nickolaychuk ◽  
Archibald McNicol
Keyword(s):  
Arachidonic Acid ◽  
Phospholipase A2 ◽  
Phospholipase C ◽  
Platelet Activation ◽  
Cytosolic Phospholipase A2 ◽  
Arachidonic Acid Release ◽  
Cytosolic Phospholipase ◽  
Low Concentrations ◽  
Acid Release ◽  
Induced Aggregation

SummaryThe release of arachidonic acid is a key component in platelet activation in response to low concentrations (1-20 g/ml) of collagen. The precise mechanism remains elusive although a variety of pathways have been implicated. In the present study the effects of inhibitors of several potentially key enzymes in these pathways have been examined. Collagen (1-10 g/ml) caused maximal platelet aggregation which was accompanied by the release of arachidonic acid, the synthesis of thromboxane A2, and p38MAPK phosphorylation. Preincubation with the dual cyclooxygenase/lipoxygenase inhibitor BW755C inhibited aggregation and thromboxane production, and reduced p38MAPK phosphorylation. A phospholipase C inhibitor, U73122, blocked collagen-induced aggregation and reduced arachidonic acid release, thromboxane synthesis and p38MAPK phosphorylation. Pretreatment with a cytosolic phospholipase A2 inhibitor, AACOCF3, blocked collagen-induced aggregation, reduced the levels of thromboxane formation and p38MAPK phosphorylation but had no significant effect on arachidonic acid release. In contrast inhibition of PKC by Rö31-8220 inhibited collagen-induced aggregation, did not affect p38MAPK phosphorylation but significantly potentiated arachidonic acid release and thromboxane formation. Collagen caused the tyrosine phosphorylation of phospholipase C 2 which was inhibited by pretreatment with U73122, unaffected by AACOCF3 and enhanced by Rö31-8220. These results suggest that cytosolic phospholipase A2 plays no role in the arachidonic acid release in response to collagen. In contrast, the data are consistent with phospholipase C 2 playing a role in an intricately controlled pathway, or multiple pathways, mediating the release of arachidonic acid in collagen-stimulated platelets.

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