Role of tyrosyl phosphorylation in neutrophil priming by tumor necrosis factor-alpha and granulocyte colony stimulating factor.
The ability of human tumor necrosis factor-alpha (TNF-alpha) 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-alpha 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 alpha-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-leucyl-phenylalanine (FMLP)-induced superoxide (O2.-) generation by TNF-alpha- or G-CSF-primed PMN. Phosphorylation of the 115-kDa protein occurred in parallel with the ligand-dependent generation of O2.-. 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-alpha.
Akimaru
K
K
Department of Medical Biology, Kochi Medical School, Japan.
Utsumi
T
T
Sato
E F
EF
Klostergaard
J
J
Inoue
M
M
Utsumi
K
K
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D000906:Antibodies; D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D019833:Genistein; D016179:Granulocyte Colony-Stimulating Factor / Q000494:pharmacology*; D006801:Humans; D066298:In Vitro Techniques; D007529:Isoflavones / Q000494:pharmacology; D007546:Isoquinolines / Q000494:pharmacology; D007700:Kinetics; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism*; D010750:Phosphoproteins / Q000097:blood / Q000302:isolation & purification; D010766:Phosphorylation; D019000:Phosphotyrosine; D010879:Piperazines / Q000494:pharmacology; D047428:Protein Kinase Inhibitors; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000097:blood*; D013481:Superoxides / Q000097:blood*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D014443:Tyrosine / Q000031:analogs & derivatives / Q000032:analysis
D000906:Antibodies; D007529:Isoflavones; D007546:Isoquinolines; D010750:Phosphoproteins; D010879:Piperazines; D047428:Protein Kinase Inhibitors; D014409:Tumor Necrosis Factor-alpha; D013481:Superoxides; D016179:Granulocyte Colony-Stimulating Factor; D019000:Phosphotyrosine; D014443:Tyrosine; D009240:N-Formylmethionine Leucyl-Phenylalanine; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D019833:Genistein; D011505:Protein-Tyrosine Kinases