Nuclear transcription factors of the NF-kappaB/Rel family are inhibited by IkappaB proteins, which inactivate NF-kappaB by trapping it in the cell cytoplasm. Phosphorylation of IkappaBs marks them out for destruction, thereby relieving their inhibitory effect on NF-kappaB. A cytokine-activated protein kinase complex, IKK (for IkappaB kinase), has now been purified that phosphorylates IkappaBs on the sites that trigger their degradation. A component of IKK was molecularly cloned and identified as a serine kinase. IKK turns out to be the long-sought-after protein kinase that mediates the critical regulatory step in NF-kappaB activation.

Recently we purified a 900 kDa cytokine-responsive IkappaB kinase complex (IKK) and molecularly cloned one of its subunits, IKKalpha, a serine kinase. We now describe the molecular cloning and characterization of IKKbeta, a second subunit of the IKK complex. IKKbeta is 50% identical to IKKalpha and like it contains a kinase domain, a leucine zipper, and a helix-loop-helix. Although IKKalpha and IKKbeta can undergo homotypic interaction, they also interact with each other and the functional IKK complex contains both subunits. The catalytic activities of both IKKalpha and IKKbeta make essential contributions to IkappaB phosphorylation and NF-kappaB activation. While the interactions between IKKalpha and IKKbeta may be mediated through their leucine zipper motifs, their helix-loop-helix motifs may be involved in interactions with essential regulatory subunits.

IkappaB [inhibitor of nuclear factor kappaB (NF-kappaB)] kinase (IKK) phosphorylates IkappaB inhibitory proteins, causing their degradation and activation of transcription factor NF-kappaB, a master activator of inflammatory responses. IKK is composed of three subunits-IKKalpha and IKKbeta, which are highly similar protein kinases, and IKKgamma, a regulatory subunit. In mammalian cells, phosphorylation of two sites at the activation loop of IKKbeta was essential for activation of IKK by tumor necrosis factor and interleukin-1. Elimination of equivalent sites in IKKalpha, however, did not interfere with IKK activation. Thus, IKKbeta, not IKKalpha, is the target for proinflammatory stimuli. Once activated, IKKbeta autophosphorylated at a carboxyl-terminal serine cluster. Such phosphorylation decreased IKK activity and may prevent prolonged activation of the inflammatory response.

It has been postulated that reactive oxygen species (ROS) may act as second messengers leading to nuclear factor (NF)-kB activation. This hypothesis is mainly based on the ®ndings that N-acetyl-L-cysteine (NAC) and pyrrolidine dithiocarbamate (PDTC), compounds recognized as potential antioxidants, can inhibit NF-kB activation in a wide variety of cell types. Here we reveal that both NAC and PDTC inhibit NF-kB activation independently of antioxidative function. NAC selectively blocks tumor necrosis factor (TNF)-induced signaling by lowering the af®nity of receptor to TNF. PDTC inhibits the IkB± ubiquitin ligase activity in the cell-free system where extracellular stimuli-regulated ROS production does not occur. Furthermore, we present evidence that endogenous ROS produced through Rac/NADPH oxidase do not mediate NF-kB signaling, but instead lower the magnitude of its activation.

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