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]P_i 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.