ENHANCED PHOSPHORYLATION OF NUCLEAR 21-KDA AND 34-KDA PROTEINS IN HEPATOMA-CELL DEATH INDUCED BY TUMOR-NECROSIS-FACTOR-ALPHA

The role of nuclear protein phosphorylation in intracellular signal tr ansduction of tumor-necrosis factor-alpha (TNF-alpha) in the human hep atoma cell line PLC(PRF/5) was investigated. TNF-alpha, which displays cytolytic activity against PLC hepatoma cells, elevated the in vitro phosphorylation of two nuclear proteins (21 kDa and 34 kDa) 16 h after treatment. The cytotoxicity and enhanced nuclear protein phosphorylat ion by TNF-alpha treatment decreased in the presence of dexamethasone. Both the 21-kDa and 34-kDa proteins were extracted with 2.2 M NaCl fr om nuclear pellets and phosphorylated in kinase reaction mixtures cont aining a high concentration of salt. By phosphoamino acid analysis, th e specificity of the nuclear kinase was found to be directed toward se rine residues. The protein kinase inhibitors H7, staurosporine and her bimycin A, inhibited the phosphorylation of the 21-kDa and 34-kDa prot eins in vitro, but calphostin C and heparin did not. The treatment of cells with 4 beta-phorbol 12-myristate 13-acetate or okadaic acid did not affect the in vitro phosphorylation of the two nuclear proteins. A n anti-Fas antibody increased the phosphorylation of the 21-kDa and 34 -kDa proteins in PLC cells. DNA fragmentation was observed in PLC cell s treated with TNF-alpha and anti-Fas antibody after 24 h treatment. T hese data suggest an involvement of nuclear protein kinase in signal-t ransduction pathways of apoptotic cell damage triggered by TNF-alpha i n PLC hepatoma cells.