The regulation by phosphorylation of 'priming' of phospholipase A2 activity in the neutrophil model system, differentiated HL60 cells.
1 Differential HL60 cells have been utilized as a model system to examine the 'priming' of neutrophil phospholipase A2 activity. In control cells activation of phospholipase A2 by a 5 min stimulation with the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (100 nM) was essentially undetectable. When cells were primed by preincubation with 5 microns cytochalasin B for 5 min arachidonate release, a measure of phospholipase A2 activation, was observed within 20 s. 2 Priming by cytochalasin B did not involve or require a change in intracellular free calcium concentration. 3 Priming was associated with an increase in general protein tyrosine phosphorylation and could also be induced by the receptor tyrosine kinase agonist granulocyte macrophage colony-stimulating factor (GM-CSF, 20 ng ml-1) and be mimicked by treatment with the phosphotyrosine phosphatase inhibitor perhydrovanadate (0.5 mM). However, increase in MAP kinase activity was not involved in the priming process. 4 Western blot analysis demonstrated that phospholipase A2 was phosphorylated in both control and primed cells, but that an increase in the amount of membrane associated enzyme was found in the primed cells. 5 Thus priming appears to be due to membrane association of the phospholipase and this may be regulated by tyrosine kinase activities.
Stewart
A
A
Institute for Cancer Studies, University of Birmingham, Medical School.
Jackson
C G
CG
Wakelam
M J
MJ
D016718:Arachidonic Acid / Q000378:metabolism; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002273:Carcinogens / Q000494:pharmacology; D002454:Cell Differentiation / Q000502:physiology; D002462:Cell Membrane / Q000201:enzymology; D003571:Cytochalasin B / Q000494:pharmacology; D004789:Enzyme Activation; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D018922:HL-60 Cells / Q000187:drug effects / Q000201:enzymology; D006801:Humans; D008246:Lysophospholipids / Q000494:pharmacology; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000187:drug effects / Q000201:enzymology*; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D010766:Phosphorylation; D010972:Platelet Activating Factor / Q000494:pharmacology; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D013268:Stimulation, Chemical; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D002273:Carcinogens; D008246:Lysophospholipids; D010972:Platelet Activating Factor; D016718:Arachidonic Acid; D003571:Cytochalasin B; D009240:N-Formylmethionine Leucyl-Phenylalanine; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D011505:Protein-Tyrosine Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D010741:Phospholipases A; D054467:Phospholipases A2; D013755:Tetradecanoylphorbol Acetate