INSULIN ACTIVATES NUCLEAR FACTOR KAPPA-B IN MAMMALIAN-CELLS THROUGH ARAF-1-MEDIATED PATHWAY

We examined the effect of insulin on nuclear factor kappa B (NF-kappa B) activity in Chinese hamster ovary (CHO) cells overexpressing wild-t ype (CHO-R cells) or -defective insulin receptors mutated at Tyr(1162) and Tyr(1163) autophosphorylation sites (CHO-Y2 cells). In CHO-R cell s, insulin caused a specific, time-, and concentration-dependent activ ation of NF-kappa B. The insulin-induced DNA-binding complex was ident ified as the p50/p65 heterodimer. Insulin activation of NF-kappa B: 1) was related to insulin receptor number and tyrosine kinase activity s ince it was markedly reduced in parental CHO cells which proved to res pond to insulin growth factor-1 and phorbol 12-myristate 13-acetate (P MA) activation, and was dramatically decreased in CHO-Y2 cells; 2) per sisted in the presence of cycloheximide and was blocked by pyrrolidine dithiocarbamate, aspirin and sodium salicylate, three compounds inter fering with I kappa B degradation and/or NF-kappa B . I kappa B comple x dissociation; 3) was independent of both PMA-sensitive and atypical (zeta) protein kinases C; and 4) was dependent on Raf-1 kinase activit y since insulin-stimulated NF-kappa B DNA binding activity was inhibit ed by 8-bromo-cAMP, a Raf-1 kinase inhibitor. Moreover, insulin activa tion of NF-kappa B-driven luciferase reporter gene expression was bloc ked in CHO-R cells expressing a Raf-1 dominant negative mutant. This i s the first evidence that insulin activates NF-kappa B in mammalian ce lls through a post-translational mechanism requiring both insulin rece ptor tyrosine kinase and Raf-1 kinase activities.