Activation of AP-1 by okadaic acid in mouse keratinocytes associated with hyperphosphorylation of c-jun.

Okadaic acid (OA), a specific inhibitor of protein phosphatases 1 and 2A, is also a potent mouse skin tumor promoter. The effects of OA on regulation of c-jun/activator protein-1 (AP-1) transcriptional activation were investigated in mouse keratinocytes. AP-1 DNA binding to the jun 12-O-tetradecanoylphorbol-13-acetate-response element (TGACATCA) as determined by gel shift analysis was strongly induced by OA (100 ng/mL) at 6 and 12 h. Preincubation of nuclear extracts with anti-c-jun antibody demonstrated that c-jun was a major component of the DNA-bound AP-1 complex induced by OA in 308 cells. Transfection of a c-jun promoter-reporter construct demonstrated that AP-1 transactivation was induced by OA. The mRNA level of the c-jun proto-oncogene was dramatically increased by 6 and 12 h of OA treatment. Furthermore, a significant induction of c-jun protein was stimulated by 6 and 12 h of OA treatment. Upon further analysis, it was found that OA induced a significant accumulation of Ser 73-phosphorylated c-jun protein in 308 cells. In summary, our data suggest that skin tumor promotion by OA is due at least in part to increased AP-1 DNA binding and transactivation mediated by c-jun hyperphosphorylation.