CYTOPLASMIC PHOSPHOLIPASE-A(2) TRANSLOCATES TO MEMBRANE-FRACTION IN HUMAN NEUTROPHILS ACTIVATED BY STIMULI THAT PHOSPHORYLATE MITOGEN-ACTIVATED PROTEIN-KINASE

The addition of the chemotactic peptide formylmethionylleucylphenylala nine (fMet-Leu-Phe) to human neutrophils pretreated with the cytokine granulocyte/macrophage colony-stimulating factor (GM-CSF) results in a 10-fold enhanced activity of phospholipase A2, measured as the releas e of arachidonic acid. It is found that GM-CSF increases the tyrosine phosphorylation, enhances the activity of a mitogen-activated protein kinase, and greatly potentiates the fMet-Leu-Phe-induced tyrosine phos phorylation and enhanced activity of this kinase. Stimuli that increas e the tyrosine phosphorylation, enhance the activity of the mitogen-ac tivated protein kinase, and cause a rise in the intracellular concentr ation of free calcium increase the amount of phospholipase A2 associat ed with the plasma membrane. This increase corresponds to a decrease i n the amount found in the cytosol. Whereas GM-CSF alone produces only a small increase in the amount of phospholipase A2 associated with the membrane, it potentiates greatly the fMet-Leu-Phe-induced increase. T he total amount (whole cell) of phospholipase A2, as measured by immun oblotting using anti-phospholipase A2 antibody, does not change upon s timulation of human neutrophils with GM-CSF, fMet-Leu-Phe, or both. In addition, the band that corresponds to phospholipase A2 is shifted up ward in membrane isolated from neutrophils stimulated with fMet-Leu-Ph e, suggesting that the enzyme has been altered, possibly phosphorylate d, though not on tyrosine residues. A working hypothesis is presented. Briefly, stimulation of human neutrophils with GM-CSF, in the absence of an additional stimulus, increases the tyrosine phosphorylation and activation of a mitogen-activated protein kinase, which in turn phosp horylates and activates cytoplasmic phospholipase A2. In the presence of an increased intracellular concentration of free calcium the phosph olipase A2 is translocated to the plasma membrane where its substrate is located. GM-CSF also potentiates greatly the fMet-Leu-Phe-induced t yrosine phosphorylation and activation of a mitogen-activated protein kinase and, since fMet-Leu-Phe causes an intracellular calcium rise, t he amount of the phospholipase A2 that is associated with the membrane fraction.