TUMOR NECROSIS FACTOR-INDUCED ACTIVATION AND INCREASED TYROSINE PHOSPHORYLATION OF MITOGEN-ACTIVATED PROTEIN (MAP) KINASE IN HUMAN FIBROBLASTS

Tumor necrosis factor (TNF) is a pleiotropic cytokine whose many demon strated actions include effects on cell growth and differentiation. TN F treatment of cells is known to lead to a rapid increase in serine/th reonine phosphorylation of many cellular proteins, but the kinases res ponsible remain largely unidentified. We show that TNF treatment induc es a rapid and transient increase in mitogen-activated protein kinase (MAPK) activity in the human diploid FS-4 cell line, for which TNF is known to be mitogenic. TNF-induced activation of MAPK was demonstrated by its enhanced ability to phosphorylate myelin basic protein in vitr o and by a characteristic shift in the electrophoretic mobility of MAP K proteins. MAPK activation was accompanied by a significant increase of MAPK phosphorylation on tyrosine residues, which was demonstrated b y P-32 labeling of cells and isolation of the labeled proteins after i mmunoprecipitation with antibodies to phosphotyrosine, and by direct i mmunoblotting of SDS-polyacrylamide gel electrophoresis-fractionated u nlabeled cell lysates with antibodies to phosphotyrosine. The pp42 and pp44 MAPK were the only proteins whose tyrosine phosphorylation was d emonstrably increased in FS-4 cells after TNF treatment. MAPK activati on is likely to represent an important component in the cascade of sig nals that link TNF receptors to various TNF-elicited cellular response s.