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Signal transduction

A regulator branches out

The cellular signalling pathway that leads to activation of the NF-κB protein has been studied for many years, and one might think that there's little left to learn. But it still has some surprises in store.

Signalling molecules usually contribute to more than one type of process in living organisms, but the NF-κB family of proteins is particularly multi-talented. It is involved in immune and inflammatory responses, in cell survival and proliferation, and in cancer — and these are just a few of its functions. The NF-κB proteins are part of a molecular cascade that starts with signals outside a cell and culminates, in the cell nucleus, with the binding of NF-κB dimers to DNA and the activation of gene expression. It had been thought that some components of this pathway work solely to regulate the movement of NF-κB to the nucleus. But on pages 655 and 659 of this issue, Yamamoto and colleagues1 and Anest and co-workers2 report a surprising new role for one such component3, an enzyme called IKK-α.

Cells must keep their NF-κB proteins on a tight leash to prevent them from activating gene transcription wantonly. So, in the absence of specific extracellular signals, the proteins are kept in the cytoplasm by inhibitors that come in two flavours: the IκB molecules, which act only as NF-κB inhibitors, and the p105 and p100 proteins, which serve both as inhibitors and as precursors of NF-κB DNA-binding subunits (Fig. 1). When cells receive appropriate cues, the IκB kinase (IKK) complex becomes active and labels the inhibitors with phosphate groups. This phosphorylation leads either to the complete degradation of the IκB inhibitors, or to the partial degradation of the p100 or p105 proteins. Free NF-κB dimers are thereby generated, which contain a DNA-binding subunit (p50 or p52) and a transcription-activating subunit, such as p65 or RelB. The dimers then move to the nucleus, where they switch on their target genes.

Figure 1: The classical NF-κB cascade, the alternative — and a new twist.
figure1

NF-κB proteins are dimers, comprising a DNA-binding subunit (such as p50 or p52) and a transcription-activating subunit (such as p65 or RelB). In cells that have not received appropriate external cues, the proteins are kept inactive either by a member of the IκB family in the classical pathway, or by an inactive precursor (in this case, p100) in the alternative pathway. In response to proteins such as tumour-necrosis factor-α (TNF-α) or lymphotoxin-β (top), the IκB kinase (IKK) complex is activated. It phosphorylates IκB and/or p100, leading to degradation of IκB and the processing of p100 into a smaller, p52 form. The p50–p65 and p52–RelB dimers (two forms of NF-κB) then move to the nucleus and activate gene expression. Yamamoto et al.1 and Anest et al.2 have found that IKK-α can itself move into the nucleus in response to TNF-α, where it associates with certain NF-κB-responsive genes and phosphorylates a histone protein, one of the components of chromatin. How it enters the nucleus and associates with the appropriate genes remains unknown. (Note that the partners of IKK-α in the complex targeted by lymphotoxin-β have not been formally identified.)

There are three known subunits in the IKK complex — two protein kinases (IKK-α and IKK-β) and a structural/regulatory subunit (NEMO/IKK-γ)3. Yamamoto et al.1 and Anest et al.2 have now identified a wholly unexpected new substrate for the IKK-α component. They show that, in response to extracellular cytokine proteins that influence the inflammatory response — such as tumour-necrosis factor-α (TNF-α) — IKK-α itself moves into the nucleus. Once there, it associates with the promoters (regulatory regions) of several NF-κB-responsive genes, and phosphorylates a component of chromatin.

Chromatin is the compact form of DNA that is found, in association with certain proteins, in the nuclei of eukaryotic organisms; its highly organized structure is important in regulating gene expression. At the heart of chromatin are the histone proteins (H1, H2A, H2B, H3 and H4), which are dynamic components of the gene-transcription machinery. Histones that are located at gene-regulatory regions undergo several types of modification that affect the expression of the corresponding genes4. For instance, the phosphorylation of histone H3 at a particular serine amino acid (serine 10) is important in inducing the transcription of so-called immediate early genes (genes that are rapidly turned on and off in response to extracellular signals)5. Yamamoto et al. and Anest et al. now show that IKK-α phosphorylates histone H3 at serine 10.

How does this fit in with what we already know about the NF-κB signalling cascade? Interestingly, it had already been suggested that the two kinases of the IKK complex have different roles. IKK-β is involved in the degradation of the IκB inhibitors in response to pro-inflammatory cytokines such as TNF-α (left-hand pathway in Fig. 1). But IKK-α, besides being involved in an NF-κB- and kinase-independent manner in regulating skin-cell differentiation6, is also thought to be part of an alternative pathway. This pathway, activated in response to specific stimuli such as lymphotoxin-β, a member of the TNF family, causes partial degradation of the p100 protein and the movement of NF-κB dimers such as p52–RelB to the nucleus (right-hand pathway in Fig. 1)3. These results have led to the suggestion that IKK-α is not involved in TNF-α-induced activation of NF-κB target genes. But other data have challenged this view7, including a report8 that it is required for TNF-α-induced expression of the gene that encodes the NF-κB inhibitor IκBα; this gene is also a target of NF-κB.

The new findings1,2 suggest that IKK-α is indeed required for TNF-α-induced gene expression — but in a non-classical way. Recruitment to the promoters of NF-κB target genes has so far been formally demonstrated only for members of the NF-κB family such as p65, the major transcription-activating subunit9,10. It has also been reported for more general transcription regulators that associate with NF-κB components10. But this is the first time that an upstream component of the signalling cascade has been shown to behave in this way.

There are a few differences between the results obtained by the two groups. Yamamoto et al.1 studied the promoter of the IκBα gene in HeLa cells, and detected IKK-α, but not IKK-β. Anest et al.2, by contrast, looked at mouse embryo fibroblast cells and found that IKK-β was recruited to the same promoter, with kinetics similar to that of IKK-α. (In fact, they also found that the third component of the complex, NEMO/IKK-γ, was recruited to that promoter, at later times.) But both groups show that IKK-β is not essential for the phosphorylation of histone H3.

These findings raise a number of questions. For instance, what protein (or proteins) conveys IKK-α to the gene promoters? The new data1,2 suggest that p65 might be responsible, although the authors did not detect a direct interaction between the two proteins. Also, does IKK-α regulate NF-κB-independent promoters? Does IKK-α-mediated phosphorylation of histone H3 regulate other histone modifications that induce gene expression? What role does IKK-β (and NEMO/IKK-γ) play when it is recruited to chromatin? Do other NF-κB-activating stimuli induce the recruitment of any of these proteins? In this context, Saccani et al.9 found that IKK-α is not recruited to the promoter of IκBα in cells stimulated with lipopolysaccharide, part of the cell wall of many bacteria — implying that such recruitment is not a general mechanism. In another seemingly contradictory result, Cao et al.11 showed that cells with an inactive version of IKK-α respond normally to TNF-α, interleukin-1 and lipopolysaccharide. But the regulation of specific NF-κB-responsive genes such as IκBα has not been studied in this genetic setting.

Whatever the answers to these questions, the discovery that IKK-α has this activity will open up a new avenue of research into the NF-κB cascade, bringing the nuclear events associated with activation of this signalling pathway back into focus.

References

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Correspondence to Alain Israël.

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