Nitric oxide represses inhibitory kB kinase through S-nitrosylation

Nitric oxide (NO) possesses anti inflammatory effects, which maybe exerted via its ability to inhibit the transcription factor, NF-kappaB. A commonly proposed mode of action for inhibition of NF-kappaB by NO involves interference with NF-kappaB binding to DNA. Because activation of inhibitory kappaB kinase (IKK), the prerequisite enzyme complex necessary to induce NF-kappaB, is subject to redox regulation, we assessed whether IKK could present a more proximal target for NO to inhibit NF-kappaB activation. We demonstrate here that S-nitroso-thiols (SNO) caused a dose-dependent inhibition of the enzymatic activity of IKK, in lung epithelial cells and in Jurkat T cells, which was associated with S-nitrosylation of the IKK complex. Using biotin derivatization of SNO, we revealed that IKKbeta, the catalytic subunit required for NF-kappaB activation, was a direct target for S-nitrosylation. A mutant version of IKKbeta containing a Cys-179-to-Ala mutation was refractory to inhibition by SNO or to increases in S-nitrosylation, in contrast to wild-type IKKbeta, demonstrating that Cys-179 is the main target for attack by SNO. Importantly, inhibition of NO synthase activity in Jurkat T cells resulted in activation of IKK, in association with its denitrosylation. Moreover, NO synthase inhibition enhanced the ability of tumor necrosis factor a to activate IKK, illustrating the importance of endogenous NO in regulating the extent of NF-kappaB activation by cytokines. Collectively, our findings demonstrate that IKKbeta is an important target for the redox regulation of NF-kappaB by endogenous or exogenous NO, providing an additional mechanism for its anti inflammatory properties.