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-coate d plastic. When PMN are kept in suspension, TNF does not induce signif icant tyrosine phosphorylation. In adherent PMN, a 42-kDa protein (p42 ) displayed the most striking increase in tyrosine phosphorylation aft er TNF stimulation. Cell lysates of TNF-stimulated PMN were separated by two-dimensional gel electrophoresis and were immunoblotted with eit her anti-phosphotyrosine (alpha-PY) mAb or anti-mitogen-activated prot ein kinase (alpha-MAPK) mAb. Both Abs detected p42, and the spots were superimposable. Cell lysates were immunoprecipitated with agarose-con jugated alpha-PY mAb, electrophoresed, and then immunoblotted with alp ha-MAPK Ab; alternatively, cell lysates were immunoprecipitated with a garose-conjugated alpha-MAPK Ab, electrophoresed, and then immunoblott ed with alpha-PY mAb. In both cases, p42 was detected. These results d emonstrate that p42 is a member of the MAPK family. TNF induces a time - and dose-dependent increase in tyrosine phosphorylation of p42 and M APK activity. The degree of p42 tyrosine phosphorylation parallels the level of MAPK activity. MAPK activity was determined by measuring P-3 2 phosphorylation of a synthetic peptide containing the recognition si te on myelin basic protein for MAPK. PMN pretreatment with genistein, a tyrosine kinase inhibitor, inhibited the TNF-induced increase in tyr osine phosphorylation and MAPK activity. These results indicate that T NF signaling involves activation of MAPK.