Extracellular signal-regulated kinase 1/2-mediated phosphorylation of JunDand FosB is required for okadaic acid-induced activator protein 1 activation

Previously, we reported that in papilloma-producing 308 mouse keratinocytes , the tumor promoter okadaic acid, a serine-threonine phosphatase inhibitor , increased binding of activator protein 1 (AP-1) to a consensus 12-O-tetra decanoylphorbol-18-acetate-responsive element (Rosenberger, S. F., and Bowd en, G.T. (1996) Oncogene 12, 2301-2308). In this study, we investigated the correlation between AP-I DNA binding and transactivation and examined mole cular mechanisms involved in this process. Using a luciferase reporter driv en by region -74 to +63 of the human collagenase gene, we demonstrated indu ction of AP-1-mediated transcription following okadaic acid treatment. By p erforming in vitro kinase assays, we found elevated activities of extracell ular signal-regulated kinase (ERH) 1/2, c-Jun N-terminal kinase, and p38 mi togen-activated protein kinase. The ERK-1/2-specific inhibitor PD 98059 com pletely abrogated okadaic acid-induced AP-1 transactivation without alterin g AP-1-expression, DNA binding, or complex composition. Phosphorylation ana lyses indicated that inhibition of ERK-1/2 decreased okadaic acid-elevated phosphorylation of JunD and FosB. To further examine the role of JunD and F osB in okadaic acid-induced AP-1 transactivation, we generated fusion prote ins of the DNA-binding domain of the yeast transcription factor Gal4 and th e transactivation domain of either JunD or FosB. Cotransfection experiments of these constructs with a Gal4-luciferase reporter demonstrated that both JunD and FosB are required for okadaic acid-induced transcription. Treatme nt with PD 98059 reduced JunD/FosB-dependent transactivation, suggesting th at ERK-1/2-mediated phosphorylation is a critical component in this process .