GRANULOCYTE-MACROPHAGE COLONY-STIMULATING FACTOR (GM-CSF) PROMOTES PHOSPHORYLATION AND AN INCREASE IN THE ACTIVITY OF CYTOSOLIC PHOSPHOLIPASE A(2) IN HUMAN NEUTROPHILS

Incubation of human neutrophils with 500 pM granulocyte-macrophage col ony-stimulating factor (GM-CSF) results in a rapid and time-dependent increase in the phosphorylation of cytosolic phospholipase A(2) (cPLA( 2)), which was reflected in a slower electrophoretic mobility of the e nzyme. The GM-CSF-induced phosphorylation of cPLA(2) was accompanied b y a parallel and time-dependent increase in the enzyme activity. Prein cubation of neutrophils with the tyrosine kinase inhibitor genistein c aused inhibition of the GM-CSF-stimulated phosphorylation and activity of cPLA(2). Immunoprecipitation of the enzyme following incubation of neutrophils with [P-32]P-i shows that cPLA(2) is phosphorylated by GM -CSF. Potato acid phosphatase caused dephosphorylation of the enzyme, indicating that cPLA(2) is indeed phosphorylated by GM-CSF. The subcel lular distribution of cPLA(2) in GM-CSF-stimulated neutrophils reveale d 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 form s of the enzyme to the membranes. Translocation of cPLA, was also achi eved after incubation with 0.1 mu M N-formylmethionyl-leucyl-phenylala nine (fMLP) after GM-CSF stimulation and when neutrophils were challen ged with the Ca2+ ionophore A23187. EDTA and EGTA were unable to solub ilize the translocated enzyme from the neutrophil membranes, indicatin g that cPLA(2) is attached to the membranes by strong bonds and not me rely due to ionic forces exerted by Ca2+. The inability of GM-CSF to p romote arachidonic acid mobilization is probably due to the fact that GM-CSF does not cause an increase in intracellular Ca2+, which is nece ssary for the translocation of the enzyme to the membranes where its s ubstrate(s) reside.