Redox regulation of nuclear factor B (NF-B) has been described, but the molecular mechanism underlying such regulation has remained unclear. We recently showed that a novel disulfide reductase, TRP14, inhibits tumor necrosis factor ␣ (TNF␣)-induced NF-B activation, and we identified the dynein light chain LC8, which interacts with the NF-B inhibitor IB␣, as a potential substrate of TRP14. We now show the molecular mechanism by which NF-B activation is redox-dependently regulated through LC8. LC8 inhibited TNF␣-induced NF-B activation in HeLa cells by interacting with IB␣ and thereby preventing its phosphorylation by IB kinase (IKK), without affecting the activity of IKK itself. TNF␣ induced the production of reactive oxygen species, which oxidized LC8 to a homodimer linked by the reversible formation of a disulfide bond between the Cys 2 residues of each subunit and thereby resulted in its dissociation from IB␣. Butylated hydroxyanisol, an antioxidant, and diphenyleneiodonium, an inhibitor of NADPH oxidase, attenuated the phosphorylation and degradation of IB␣ by TNF␣ stimulation. In addition LC8 inhibited NF-B activation by other stimuli including interleukin-1␤ and lipopolysaccharide, both of which generated reactive oxygen species. Furthermore, TRP14 catalyzed reduction of oxidized LC8. Together, our results indicate that LC8 binds IB␣ in a redoxdependent manner and thereby prevents its phosphorylation by IKK. TRP14 contributes to this inhibitory activity by maintaining LC8 in a reduced state.

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