TNF-alpha induces tyrosine phosphorylation of mitogen-activated protein kinase in adherent human neutrophils.
Recombinant human TNF-alpha induces increased tyrosine phosphorylation of several proteins in human neutrophils (PMN) adhered to serum-coated plastic. When PMN are kept in suspension, TNF does not induce significant tyrosine phosphorylation. In adherent PMN, a 42-kDa protein (p42) displayed the most striking increase in tyrosine phosphorylation after TNF stimulation. Cell lysates of TNF-stimulated PMN were separated by two-dimensional gel electrophoresis and were immunoblotted with either anti-phosphotyrosine (alpha-PY) mAb or anti-mitogen-activated protein kinase (alpha-MAPK) mAb. Both Abs detected p42, and the spots were superimposable. Cell lysates were immunoprecipitated with agarose-conjugated alpha-PY mAb, electrophoresed, and then immunoblotted with alpha-MAPK Ab; alternatively, cell lysates were immunoprecipitated with agarose-conjugated alpha-MAPK Ab, electrophoresed, and then immunoblotted with alpha-PY mAb. In both cases, p42 was detected. These results demonstrate that p42 is a member of the MAPK family. TNF induces a time-and dose-dependent increase in tyrosine phosphorylation of p42 and MAPK activity. The degree of p42 tyrosine phosphorylation parallels the level of MAPK activity. MAPK activity was determined by measuring 32P phosphorylation of a synthetic peptide containing the recognition site on myelin basic protein for MAPK. PMN pretreatment with genistein, a tyrosine kinase inhibitor, inhibited the TNF-induced increase in tyrosine phosphorylation and MAPK activity. These results indicate that TNF signaling involves activation of MAPK.
Rafiee
P
P
Division of Pulmonary and Critical Care Medicine, Stanford University Medical Center, CA 94305, USA.
Lee
J K
JK
Leung
C C
CC
Raffin
T A
TA
D000595:Amino Acid Sequence; D002448:Cell Adhesion / Q000502:physiology; D002478:Cells, Cultured; D004789:Enzyme Activation / Q000502:physiology; D019833:Genistein; D006801:Humans; D015151:Immunoblotting; D007529:Isoflavones / Q000494:pharmacology; D008969:Molecular Sequence Data; D009504:Neutrophils / Q000201:enzymology*; D011233:Precipitin Tests; D011494:Protein Kinases / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000502:physiology*
D007529:Isoflavones; D014409:Tumor Necrosis Factor-alpha; D019833:Genistein; D011494:Protein Kinases; D011505:Protein-Tyrosine Kinases