Role of tyrosyl phosphorylation in neutrophil priming by tumor necrosis factor-α and granulocyte colony stimulating factor

Abstract

The ability of human tumor necrosis factor-α (TNF-α) and human granulocyte colony stimulating factor (G-CSF) to induce phosphorylation of protein tyrosyl residues in human peripheral neutrophils (PMN) was investigated by Western blot analysis with antiphosphotyrosine antibody. Both TNF-α and G-CSF increased the tyrosyl phosphorylation of various proteins, such as species of 54-, 63-, 72-, 83-, 98-, 108-, and 115-kDa proteins. The ligand-stimulated tyrosyl phosphorylation of the 115-kDa protein was time- and concentration-dependent. When the 115-kDa protein was phosphorylated, it was recovered from membrane fractions. The phosphorylation of the 115-kDa protein was inhibited by genistein and α-cyano-3-ethoxy-4-hydroxy-5-phenylthiomethylcinnamamide (ST 638), inhibitors of tyrosine kinase (TK), and was enhanced by 1-(5-isoquinoline-sulfonyl) methyl-piperazine dihydrochloride (H-7) and staurosporine, inhibitors of Ca 2+- and phospholipid-dependent protein kinase (PKC). Similar inhibition by the TK inhibitors and stimulation by the PKC inhibitors were also observed with formylmethionyl-leucylphenylalanine (FMLP)-induced superoxide (O ⨪2) generation by TNF-α- or G-CSF-primed PMN. Phosphorylation of the 115-kDa protein occurred in parallel with the ligand-dependent generation of O ⨪2. These and other observations suggested that substrate proteins for tyrosine kinase, such as the 115-kDa protein, might play critical roles in the mechanism for priming of neutrophils. This is the first report describing that tyrosyl phosphorylation is involved in the priming of neutrophils by G-CSF and TNF-α.