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 transduction of tumor-necrosis factor-alpha (TNF-alpha) in the human hepatoma 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 phosphorylation 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 from nuclear pellets and phosphorylated in kinase reaction mixtures containing a high concentration of salt. By phosphoamino acid analysis, the specificity of the nuclear kinase was found to be directed toward serine residues. The protein kinase inhibitors H7, staurosporine and herbimycin A, inhibited the phosphorylation of the 21-kDa and 34-kDa proteins 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. An anti-Fas antibody increased the phosphorylation of the 21-kDa and 34-kDa proteins in PLC cells. DNA fragmentation was observed in PLC cells treated with TNF-alpha and anti-Fas antibody after 24 h treatment. These data suggest an involvement of nuclear protein kinase in signal-transduction pathways of apoptotic cell damage triggered by TNF-alpha in PLC hepatoma cells.
Masumi-Fukazawa
A
A
Department of Safety Research on Biologics, National Institute of Health, Tokyo, Japan.
Kohase
M
M
Fuse
A
A
D017209:Apoptosis / Q000187:drug effects*; D003907:Dexamethasone / Q000494:pharmacology; D004336:Drug Antagonism; D006801:Humans; D009687:Nuclear Proteins / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D014407:Tumor Cells, Cultured / Q000187:drug effects; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D009687:Nuclear Proteins; D014409:Tumor Necrosis Factor-alpha; D003907:Dexamethasone