Abstract
Redox regulation of nuclear factor kappaB (NF-kappaB) 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 (TNFalpha)-induced NF-kappaB activation, and we identified the dynein light chain LC8, which interacts with the NF-kappaB inhibitor IkappaBalpha, as a potential substrate of TRP14. We now show the molecular mechanism by which NF-kappaB activation is redox-dependently regulated through LC8. LC8 inhibited TNFalpha-induced NF-kappaB activation in HeLa cells by interacting with IkappaBalpha and thereby preventing its phosphorylation by IkappaB kinase (IKK), without affecting the activity of IKK itself. TNFalpha 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 IkappaBalpha. Butylated hydroxyanisol, an antioxidant, and diphenyleneiodonium, an inhibitor of NADPH oxidase, attenuated the phosphorylation and degradation of IkappaBalpha by TNFalpha stimulation. In addition LC8 inhibited NF-kappaB activation by other stimuli including interleukin-1beta and lipopolysaccharide, both of which generated reactive oxygen species. Furthermore, TRP14 catalyzed reduction of oxidized LC8. Together, our results indicate that LC8 binds IkappaBalpha 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.
Highlights
We recently showed that a novel disulfide reductase, TRP14, inhibits TNF␣-induced nuclear factor B (NF-B) activation by suppressing the phosphorylation of IB␣, and we identified LC8 as a potential substrate of TRP14 [28]
Reagents and Antibodies—LPS, butylated hydroxyanisol (BHA), diphenyleneiodonium (DPI), NADPH, and dithiothreitol (DTT) were obtained from Sigma; 4Ј,6-diamidino-2-phenylindole and 5-(and-6-)chloromethyl-2Ј,7Ј-dichlorodihydrofluorescein diacetate (CM-H2DCFDA), acetyl ester were from Molecular Probes; MG132 was from Calbiochem; glutathione-Sepharose and nickel-chelating Sepharose were from Amersham Biosciences; and TNF␣ and IL-1 were from R & D Systems
LC8 Expression Inhibits TNF␣-induced NF-B Activation— Given that LC8 inhibited IKK-mediated phosphorylation of IB␣ in vitro, we investigated the effect of forced expression of LC8 on the NF-B signaling pathway in HeLa cells
Summary
Reagents and Antibodies—LPS, butylated hydroxyanisol (BHA), diphenyleneiodonium (DPI), NADPH, and dithiothreitol (DTT) were obtained from Sigma; 4Ј,6-diamidino-2-phenylindole and 5-(and-6-)chloromethyl-2Ј,7Ј-dichlorodihydrofluorescein diacetate (CM-H2DCFDA), acetyl ester were from Molecular Probes; MG132 was from Calbiochem; glutathione-Sepharose and nickel-chelating Sepharose were from Amersham Biosciences; and TNF␣ and IL-1 were from R & D Systems. After the removal of debris by centrifugation, the remaining soluble fraction was applied at a flow rate of 2 ml/min to a DEAE-Sepharose column that had been equilibrated with a solution containing 20 mM Tris-HCl (pH 8.0) and 1 mM EDTA. For the bacterial expression of IB␣, a DNA fragment encoding human IB␣ was amplified by PCR from HeLa cell cDNA and cloned into the NdeI and BamHI sites of pET14b. In Vitro Kinase Assay—The phosphorylation of IB␣ (1 g) was performed for 30 min at 30 °C with an IKK immune complex in a final volume of 40 l containing 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 5 mM DTT, 1 mM MgCl2, 1 mM Na3VO4, 5 mM glycerophosphate, 100 M ATP, and 10 Ci of [␥-32P]ATP. AMS and N-ethylmaleimide were used to mask free thiol groups
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