AP-1 TRANSCRIPTIONAL ACTIVITY IS REGULATED BY A DIRECT ASSOCIATION BETWEEN THIOREDOXIN AND REF-1

Thioredoxin (TRX) is a pleiotropic cellular factor that has thiol-medi ated redox activity and is important in regulation of cellular process es, including proliferation, apoptosis, and gene expression. The activ ity of several transcription factors is posttranslationally altered by redox modification(s) of specific cysteine residue(s). One such facto r is nuclear factor (NF)-kappa B, whose DNA-binding activity is marked ly augmented by TRX treatment irt vitro. Similarly, the DNA-binding ac tivity of activator protein 1 (AP-1) is modified by a DNA repair enzym e, redox factor 1 (Ref-1), which is identical to a DNA repair enzyme, AP endonuclease. Ref-1 activity is in turn modulated by various redox- active compounds, including TRX. We here report the molecular cascade of redox regulation of AP-1 mediated by TRX and Ref-1. Phorbol 12-myri state 13 acetate efficiently translocated TRX into the HeLa cell nucle us where Ref-1 preexists. This process seems to be essential for AP-1 activation by redox modification because co-overexpression of TRX and Ref-1 in COS-7 cells potentiated AP-1 activity only after TRX,vas tran sported into the nucleus by phorbol 12-myristate 13 acetate treatment. To prove the direct active site-mediated association between TRX and Ref-1, we generated a series of substitution-mutant cysteine residues of TRX. In both an in vitro diamide-induced cross-linking study and an in vivo mammalian two-hybrid assay we proved that TRX can associate d irectly with Ref-1 in the nucleus; also, we demonstrated the requireme nt of cysteine residues in the TRX catalytic center for the potentiati on of AP-1 activity. This report presents an example of a cascade in c ellular redox regulation.