Granulocyte-macrophage colony-stimulating factor (GM-CSF) promotes phosphorylation and an increase in the activity of cytosolic phospholipase A2 in human neutrophils.
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 microM N-formylmethionyl-leucyl-phenyl-alanine (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.
Nahas
N
N
Department of Physiology, University of Connecticut Health Center, Farmington 06030-3505, USA.
Waterman
W H
WH
Sha'afi
R I
RI
D001692:Biological Transport; D002384:Catalysis; D002462:Cell Membrane / Q000201:enzymology; D003600:Cytosol / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019833:Genistein; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology*; D006801:Humans; D066298:In Vitro Techniques; D007529:Isoflavones / Q000494:pharmacology; D007700:Kinetics; D009504:Neutrophils / Q000201:enzymology*; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D010766:Phosphorylation; D011233:Precipitin Tests
D004791:Enzyme Inhibitors; D007529:Isoflavones; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D019833:Genistein; D010741:Phospholipases A; D054467:Phospholipases A2