Abstract
The processing of the nfkappab2 gene product p100 to generate p52 is a regulated event, which is important for the instrumental function of NF-kappaB. We previously demonstrated that this tightly controlled event is regulated positively by NF-kappaB-inducing kinase (NIK) and its downstream kinase, IkappaB kinase alpha (IKKalpha). However, the precise mechanisms by which NIK and IKKalpha induce p100 processing remain unclear. Here, we show that, besides activating IKKalpha, NIK also serves as a docking molecule recruiting IKKalpha to p100. This novel function of NIK requires two specific amino acid residues, serine 866 and serine 870, of p100 that are known to be essential for inducible processing of p100. We also show that, after being recruited into p100 complex, activated IKKalpha phosphorylates specific serines located in both N- and C-terminal regions of p100 (serines 99, 108, 115, 123, and 872). The phosphorylation of these specific serines is the prerequisite for ubiquitination and subsequent processing of p100 mediated by the beta-TrCP ubiquitin ligase and 26 S proteasome, respectively. These results highlight the critical but different roles of NIK and IKKalpha in regulating p100 processing and shed light on the mechanisms mediating the tight control of p100 processing. These data also provide the first evidence for explaining why overexpression of IKKalpha or its activation by many other stimuli such as tumor necrosis factor and mitogens fails to induce p100 processing.
Highlights
The transcription factor NF-B plays a central role in the regulation of diverse biological processes including immune response, development, cell growth, and survival [1,2,3,4]
Because NF-Binducing kinase (NIK) induces the catalytic activity of IKK␣ [37], we examined whether the induction of IKK␣/p100 binding by NIK is the result of activation of IKK␣
The proper activation of this NF-B pathway plays a crucial role in the development, organization, and function of lymphoid tissue [7], because those immune defects are shared by all mutant mice with genetic defects in this pathway such as those deficient in NIK [25], lymphotoxin and lymphotoxin  receptor [39, 40], B-cell-activating factor and B-cell-activating factor receptor [26, 41], and NF-B2 [22, 23]
Summary
The transcription factor NF-B plays a central role in the regulation of diverse biological processes including immune response, development, cell growth, and survival [1,2,3,4]. The canonical pathway of NF-B activation involves inducible IB degradation, which can be stimulated by various cellular stimuli, such as T-cell mitogens, proinflammatory cytokines, and antigens These stimuli trigger an IB kinase (IKK) complex, which consists of IB kinase ␣ (IKK␣), IKK (two catalytic subunits), and IKK␥ (regulatory subunit, named NEMO), to phosphorylate specific serines within the IB sequence. The NIK/IKK␣-specific NF-B pathway cannot be stimulated by most of the classical NF-B inducers but rather respond to signals involved in B-cell maturation and lymphoid organogenesis including those triggered by lymphotoxin  [15, 18], B-cell-activating factor [19, 20], and CD40 ligand [21]. This “docking” function of NIK requires the C-terminal serines, Ser-866 and Ser-870, of p100 Both the N- and C-terminal regions of p100 contain IKK␣ phosphorylation sites. These IKK␣ phosphorylation sites together with the IKK␣ docking sites are essential for NIK-induced ubiquitination and processing of p100
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