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TNF-alpha induces tyrosine phosphorylation of mitogen-activated protein kinase in adherent human neutrophils
1Division of Pulmonary and Critical Care Medicine, Stanford University Medical Center, CA 94305, USA.
Abstract:
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.
Insights
Tumor Necrosis Factor-alpha (TNF) activates mitogen-activated protein kinase (MAPK) in adhered human neutrophils. This involves increased tyrosine phosphorylation of MAPK, a key signaling pathway.
Area of Science:
- Immunology
- Cell Biology
- Signal Transduction
Background:
- Human neutrophils (PMN) play critical roles in immune responses.
- Tumor Necrosis Factor-alpha (TNF) is a key cytokine involved in inflammation and immunity.
- Cell adhesion is known to modulate cellular signaling pathways.
Purpose of the Study:
- To investigate the effect of TNF-alpha on protein tyrosine phosphorylation in human neutrophils.
- To identify the specific signaling proteins activated by TNF-alpha in adhered neutrophils.
- To elucidate the role of mitogen-activated protein kinase (MAPK) in TNF-alpha signaling.
Main Methods:
- Human neutrophils were stimulated with recombinant human TNF-alpha.
- Protein tyrosine phosphorylation was assessed using 2D gel electrophoresis and Western blotting with anti-phosphotyrosine (alpha-PY) and anti-mitogen-activated protein kinase (alpha-MAPK) antibodies.
- MAPK activity was measured by 32P phosphorylation of a myelin basic protein peptide.
- Tyrosine kinase inhibitor genistein was used to assess the role of tyrosine kinases.
Main Results:
- TNF-alpha induced significant tyrosine phosphorylation of several proteins, notably a 42-kDa protein (p42), in adhered neutrophils, but not in suspended cells.
- p42 was identified as a member of the MAPK family.
- TNF-alpha stimulation resulted in a time- and dose-dependent increase in p42 tyrosine phosphorylation and MAPK activity.
- Inhibition of tyrosine kinases by genistein blocked TNF-alpha-induced tyrosine phosphorylation and MAPK activation.
Conclusions:
- TNF-alpha signaling in adhered human neutrophils involves the activation of MAPK.
- The activation of MAPK by TNF-alpha is dependent on tyrosine kinase activity.
- p42 is a crucial substrate in the TNF-alpha-mediated MAPK activation pathway in neutrophils.