Tumor necrosis factor-induced protein phosphorylation in human neutrophils.
Protein phosphorylation is central to multiple regulatory processes in cells. Tumor necrosis factor (TNF), a cytokine synthesized by macrophages, effects polymorphonuclear leukocyte (neutrophil) chemotaxis, induces superoxide anion generation, and mediates neutrophil adhesion to endothelial cells. Although protein phosphorylation is almost certainly involved in many TNF-mediated neutrophil functions, little is known about TNF's impact on neutrophil protein phosphorylation. Therefore, we studied human recombinant TNF-alpha-induced protein phosphorylation in human neutrophils. Neutrophils were preincubated with 32PO(4)2- and treated with a variety of stimulatory agents. One- and two-dimensional polyacrylamide gel electrophoresis was used to analyze phosphorylated proteins. Phosphoaminoacids were identified by two-dimensional thin layer chromatography electrophoresis. The findings were as follows: (1) TNF induces the phosphorylation of two 16-kD proteins (pI = 5.9 and 6.1) by 5- to 6-fold, and a 57-kD protein (pI = 5.8) by 3- to 4-fold compared with untreated neutrophils; (2) these proteins are phosphorylated as early as 15 min after stimulation with TNF, and phosphorylation is induced by concentrations of TNF as low as 1 ng/ml (10 U/ml); (3) TNF induces the phosphorylation of proteins at either serine or threonine residues and not at tyrosine; (4) TNF-stimulated neutrophils show a unique pattern of protein phosphorylation when compared to neutrophils treated with formylmethionylleucylphenylalanine; (5) lipopolysaccharide does not induce protein phosphorylation in neutrophils; (6) a 16-kD protein is phosphorylated in response to TNF in neutrophils but not in mononuclear cells; and (7) protein kinase inhibitors appear to have no effect on TNF-induced protein phosphorylation. Thus, the mechanism of action of TNF on neutrophils may involve protein phosphorylation.
Crowley
J J
JJ
Department of Medicine, Stanford University School of Medicine, California 94305-5236.
Raffin
T A
TA
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D002478:Cells, Cultured; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D066298:In Vitro Techniques; D007526:Isoelectric Point; D007546:Isoquinolines / Q000494:pharmacology; D008970:Molecular Weight; D009504:Neutrophils / Q000378:metabolism*; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D010769:Phosphothreonine / Q000378:metabolism; D010879:Piperazines / Q000494:pharmacology; D047428:Protein Kinase Inhibitors; D011994:Recombinant Proteins; D013449:Sulfonamides / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D007546:Isoquinolines; D010750:Phosphoproteins; D010879:Piperazines; D047428:Protein Kinase Inhibitors; D011994:Recombinant Proteins; D013449:Sulfonamides; D014409:Tumor Necrosis Factor-alpha; D010769:Phosphothreonine; D010768:Phosphoserine; C017967:W 7; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine