Dynein light chain LC8 negatively regulates NF-κB through the redox-dependent interaction with IκBα

Redox regulation of nuclear factor kappa B (NF-kappa 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 alpha (TNF alpha)-induced NF-kappa B activation, and we identified the dynein light chain LC8, which interacts with the NF-kappa B inhibitor I kappa B alpha, as a potential substrate of TRP14. We now show the molecular mechanism by which NF-kappa B activation is redox-dependently regulated through LC8. LC8 inhibited TNF alpha-induced NF-kappa B activation in HeLa cells by interacting with I kappa B alpha and thereby preventing its phosphorylation by I kappa B kinase (IKK), without affecting the activity of IKK itself. TNF alpha 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 I kappa B alpha. Butylated hydroxyanisol, an antioxidant, and diphenyleneiodonium, an inhibitor of NADPH oxidase, attenuated the phosphorylation and degradation of I kappa B alpha by TNF alpha stimulation. In addition LC8 inhibited NF-kappa B activation by other stimuli including interleukin-1 beta and lipopolysaccharide, both of which generated reactive oxygen species. Furthermore, TRP14 catalyzed reduction of oxidized LC8. Together, our results indicate that LC8 binds I kappa B alpha in a redox-dependent manner and thereby prevents its phosphorylation by IKK. TRP14 contributes to this inhibitory activity by maintaining LC8 in a reduced state.