Induction of p100 Processing by NF- (cid:1) B-inducing Kinase Involves Docking I (cid:1) B Kinase (cid:2) (IKK (cid:2) ) to p100 and IKK (cid:2) -mediated Phosphorylation*

The processing of the nf (cid:1) b2 gene product p100 to generate p52 is a regulated event, which is important for the instrumental function of NF- (cid:1) B. We previously demonstrated that this tightly controlled event is regulated positively by NF- (cid:1) B-inducing kinase (NIK) and its downstream kinase, I (cid:1) B kinase (cid:2) (IKK (cid:2) ). However, the pre-cise mechanisms by which NIK and IKK (cid:2) induce p100 processing remain unclear. Here, we show that, besides activating IKK (cid:2) , NIK also serves as a docking molecule recruiting IKK (cid:2) 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 IKK (cid:2) phos-phorylates 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 -TrCP ubiquitin ligase and 26 S proteasome, respectively. These highlight the critical but different roles of NIK and IKK (cid:2) 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 or by as and kinase; GST, glutathione S -transferase; ARD, ankyrin repeat domain; HRP, horseradish peroxidase; HA, hemagglutinin; IB, immunoblotting; co-IP, co-immunoprecipitation; IP, immunoprecipitation; KA, kinase-in-active; (cid:3) -TrCP, (cid:3) -transducin repeat-containing protein.

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). Consistently, deregulated function of NF-B contributes to the development of various cell malignancies (5)(6)(7). NF-B represents a family of related DNA-binding proteins, which in mammals includes five members: RelA (p65); RelB; c-Rel; p50; and p52 (8). The NF-B proteins primarily form p50/Rel or p52/Rel heterodimers, although they also may function as various other homodimers and heterodimers (8). The NF-B dimers are sequestered normally in the cytoplasm by ankyrin repeat-containing inhibitors called IB proteins (1). 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) 1 complex, which consists of IB kinase ␣ (IKK␣), IKK␤ (two catalytic subunits), and IKK␥ (regulatory subunit, also named NEMO), to phosphorylate specific serines within the IB sequence. The phosphorylated IB then is targeted for ubiquitination and proteasome-mediated degradation, allowing the NF-B dimers to move to the nucleus and transactivate target genes (4,7).
Unlike the Rel proteins, p50 and p52 are synthesized as large precursors such as NF-B1 p105 and NF-B2 p100, respectively (8 -10). These precursor proteins generate the mature p50 and p52 NF-B subunits through proteasome-mediated processing, which involves selective degradation of their Cterminal portions (3,13). Interestingly, the C-terminal regions of both p105 and p100 contain ankyrin repeats and function as IB-like inhibitors of NF-B (11,12). Thus, the processing of p105 and p100 not only serves to generate p50 and p52 but also plays a role in liberating specific NF-B complexes such as the RelB-containing complexes. Whereas the processing of p105 is constitutive and largely cotranslational (14), the processing of p100 is tightly regulated through its inducible phosphorylation and polyubiquitination (15). The induction of p100 phosphorylation and subsequent processing are mediated by the NF-Binducing kinase (NIK) and its downstream kinase IKK␣ (15,16). Interestingly, neither IKK␤ nor IKK␥ is required for this non-canonical NF-B pathway, although both IKK␤ and IKK␥ are essential for the canonical NF-B activation (16,17). Consistently, 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).
The tightly regulated p100 processing may be important for proper regulation of NF-B function in cell growth and survival. Recent gene targeting studies indicated that the germ line knock-out of nfb2 gene with no expression of both p100 and p52 results in severe defects in B-cell function and impairment in the formation of proper architecture in peripheral lymphoid organs (22,23), a phenotype not observed in nfb1-deficient mice (24). Similarly, mice having defects in p100 processing, such as the alymphoplasia mice (carrying nik gene mutation) (for details see Ref. 25) and the A/WySnJ mice (carrying mutations in the B-cell-activating factor receptor gene) (for details see Ref. 26), also show similar phenotypes. Conversely, overexpression of p52 in the absence of p100 in p100 knock-in mice leads to lymphocyte hyperplasia and transformation (27). In humans, chromosomal translocations that cause nfb2 gene rearrangement, leading to constitutive processing of p100, are associated with the development of various lymphomas (5, 7, 15, 28 -30). Interestingly, deregulated p100 processing also is found to be associated with T-cell transformation by the human T-cell leukemia virus type 1 and many other human cancers (7,17,31,32).
Because proper processing of p100 plays an essential role in the development and maturation of lymphoid organs, whereas deregulated p100 processing contributes to human malignancies, it is very important to define the detailed mechanisms of p100 processing. In this study, we have demonstrated that NIK not only activates its downstream kinase IKK␣ but also recruits IKK␣ into the p100 complex. This "docking" function of NIK requires the C-terminal serines, Ser-866 and Ser-870, of p100. Interestingly, 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.
Immunoblotting (IB) and Coimmunoprecipitation (Co-IP)-293 cells were transfected with the indicated expression vectors using DEAEdextran and lysed in radioimmunoprecipitation assay buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.25% sodium deoxycholate, 1% Nonidet P-40, 1 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride) supplemented with a protease inhibitor mixture followed by IB or co-IP assays as described previously (15,17). The amounts of cell lysates were ϳ7 g for IB and 250 g for co-IP assays.
In Vitro Kinase Assay-In vitro kinase assays were performed as described previously (16,17). Purified recombinant IKK␣ and IKK␤ proteins (15 ng, gifts from Dr. M. Karin) were incubated at 30°C for 20 min in a kinase buffer (20 mM Hepes, pH 7.6, 20 mM MgCl 2 , 20 mM ␤-glycerophosphate, 1 mM EDTA, and 2 mM dithiothreitol) containing [␥-32 P]dATP and labeled substrates. The phosphorylated proteins were fractionated by SDS-PAGE, transferred onto nitrocellulose membranes, and visualized by autoradiography. The membrane was used subsequently for IB to analyze the protein level of substrates.
In Vivo Ubiquitin Conjugation Assays-293 cells were transfected with HA-tagged ubiquitin and p100 or its mutants in the presence or absence of NIK. After 36 -48 h post-transfection, the cells were lysed in radioimmunoprecipitation assay buffer and immediately subjected to immunoprecipitation (IP) using anti-p100 antibody. The agarose beads were washed three times with radioimmunoprecipitation assay buffer followed by two additional washes with radioimmunoprecipitation assay buffer supplemented with 1 M urea. The eluted ubiquitin-conjugated p100 by SDS loading buffer was analyzed by IB using anti-HA-HRP (16,17).

RESULTS
NIK Promotes the Binding of IKK␣ to p100 -As described earlier (15,17), overexpressed IKK␣ or its activation by various stimuli that trigger the canonical NF-B pathway fails to induce productive processing of p100. However, the expression of a very low amount of NIK could strongly induce the processing of p100. These findings suggest that besides activating the catalytic activity of IKK␣, NIK may exert additional functions in the induction of p100 processing. In this regard, our previous studies indicated that NIK but not IKK␣ could interact efficiently with p100, although IKK␣ could associate physically with NIK (15,33). Thus, we predicted that NIK might target IKK␣ into the p100 complex. To test this possibility, we performed co-IP assays. As expected, no significant binding between p100 and IKK␣ could be detected when these two proteins were coexpressed in 293 cells (Fig. 1A, top panel, lane 2). Interestingly, however, the two proteins became associated stably when NIK also was expressed in cells (lane 3). Because NIK induces the catalytic activity of IKK␣ (37), we examined FIG. 1. NIK recruitment of IKK␣ into p100 is independent of activity of both IKK␣ and NIK. A, induction of p100/IKK␣ interaction by NIK is independent on the kinase activity of IKK␣. 293 cells were transfected with expression vectors encoding p100 (0.4 g) and HA-tagged IKK␣ (0.25 g) or its mutants either in the absence (Ϫ) or presence (ϩ) of HA-tagged NIK (0.25 g). The cell lysates were subjected to immunoprecipitation using anti-p100N, and the coprecipitated IKK␣ was detected by immunoblotting using anti-HA-HRP (top panel). Some of cell lysates were subjected directly to immunoblotting to monitor the expression levels of HA-tagged IKK␣ and IKK␣ mutants (middle panel) as well as the p100 and its processing product p52 (bottom panel). Wild type (WT), SS/EE, and SS/AA indicated in figure are wild type, dominant-active, and dominant-negative forms of IKK␣, respectively. B, the kinase activity of NIK is dispensable for its recruitment of IKK␣ into p100. 293 cells were transfected with indicated constructs followed by analyzing the p100/IKK␣ interaction (top panel), expression levels of IKK␣ and NIKs (middle panel), and p100 processing (bottom panel) as described in A. WT, KA, and alymphoplasia in this figure are abbreviations for NIK wild type, kinase-dead, and glycine 855 to arginine point mutants, respectively, whereas SS/EE is the constitutively activated form of IKK␣.
whether the induction of IKK␣/p100 binding by NIK is the result of activation of IKK␣. The co-IP studies were performed using dominant-active (SS/EE) and kinase-inactive (SS/AA) forms of IKK␣. In the absence of NIK, neither the IKK␣ SS/EE nor the IKK␣ SS/AA could interact significantly with p100 (lanes 4 and 6). On the other hand, both of the IKK mutants formed a stable complex with p100 in the presence of NIK (lanes 5 and 7). Thus, the NIK-induced recruitment of IKK␣ to p100 is independent of the catalytic activity of IKK␣. This finding provides an explanation for the low efficiency of IKK␣ SS/EE in inducing p100 processing (Fig. 1A, bottom panel, lane 4).
We then investigated whether the recruitment of IKK␣ to p100 requires the kinase activity of NIK. When coexpressed with IKK␣ and p100, both the wild type NIK and its kinaseinactive form (NIK KA) could promote the binding of IKK␣ to p100 (Fig. 1B, upper panel, lanes 3 and 5), although the NIK KA failed to induce p100 processing (15). Together with the results presented in Fig. 1A, these observations suggest that NIK promotes the IKK␣/p100 binding via a physical mechanism rather than via catalytic activation of IKK␣. In this regard, prior studies have shown that the alymphoplasia mutation of NIK diminishes its physical interaction with IKK␣ or p100, although it does not affect its catalytic activity (15,38). Interestingly, the NIK alymphoplasia mutant also exhibited reduced activity in promoting the IKK␣/p100 association ( suggesting that NIK has an additional function besides activating and recruiting IKK␣ into p100, which is also required for inducible p100 processing. Nevertheless, these results clearly indicate that the ability of NIK to bind to IKK␣ and p100 is essential for recruiting IKK␣ to p100 and that this novel function of NIK does not require the catalytic activity of NIK or IKK␣. Serines 866 and 870 of p100 Serve as a Docking Site for NIK-induced IKK␣ Binding-We have previously shown that two C-terminal serines, Ser-866 and Ser-870, of p100 are essential for NIK-induced p100 processing (15). Although these two serines are located in the IKK␣ phosphorylation site of p100, it remains unclear whether they serve as the direct target of IKK␣. We examined whether these two critical residues are involved in the binding of p100 by IKK␣. As expected, the wild type p100 formed a stable complex with IKK␣ in the presence of NIK ( Fig. 2A, top panel, lane 2). In contrast, the p100 mutant harboring serine to alanine substitutions at serines 866 and 870 (p100 S866A/S870A) was largely defective in the inducible binding to IKK␣ (lane 4). The failure of p100 S866A/S870A to recruit IKK␣ was not attributed to its inability in binding NIK, because a similar level of NIK binding activity was detected with wild type p100 and p100 S866A/S870A (Fig.  2B, top panel, lanes 2 and 3). The expression levels of these two different p100 constructs as well as NIK in the different cells were also comparable (middle and bottom panels). These data strongly imply that serines 866 and 870 may function as a docking site for NIK-induced IKK␣ binding.
Serine 872, but Not Serines 866 and 870, of p100 Serves as the IKK␣ Phosphorylation Site-Our previous studies have indicated that serines 866, 870, and 872 of p100 are candidates for IKK␣ phosphorylation sites, because mutation of these serines abolishes the phosphorylation of the C-terminal region of p100 by I⌲⌲␣ (16,17). However, it has remained unclear whether all of the three serines or just some of them are the IKK␣ targets. To answer this question, GST-p100C mutants harboring serine to alanine substitutions at serines 866 and 870 (S866A/S870A), serine 872 (S872A), or all three serines (SSS/AAA) were examined for phosphorylation by IKK␣. As expected, purified recombinant IKK␣ efficiently phosphorylated wild type GST-p100C but not the GST-p100C SSS/AAA (Fig. 3A, upper panel, lanes 1 and 2). Surprisingly, mutation of serines 866 and 870 did not affect the p100 phosphorylation by IKK␣ (lane 3). On the other hand, the p100C mutant harboring serine 872 mutation (GST-p100C S872A) completely lost its ability to be phosphorylated by IKK␣ (lane 4). Thus, serine 872, but not serines 866 and 870, serves as a target of IKK␣. Taken together with the data presented in Fig. 2, these findings suggest that serines 866 and 870 are required for the recruitment of IKK␣ to p100 but are not the actual phosphorylation sites for IKK␣.
To address the role of serine 872 in the inducible processing of p100, wild type p100 or p100 mutant harboring this serine mutation (p100 S872A) was transfected into 293 cells either in the absence or presence of the p100-processing inducer, NIK. Compared with the wild type p100, the p100 S872A exhibited a dramatic defect in NIK-induced processing (Fig. 3B, upper  panel, lanes 2 and 4). Interestingly, the mutation of serine 872 to a phosphomimetic residue (aspartic acid) generated a p100 mutant (p100 S872D) that exhibited a higher inducible processing ability than p100 wild type (lane 6). In contrast, the p100 mutant harboring serine to aspartic acid substitutions at serines 866 and 870 (p100 S866A/S870D) was defective in NIK-induced processing (lane 8), a phenotype similar to that of p100 S866A/S870A (lane 10) (15). These results further suggested that serine 872, but not serine 866 or 870, is an IKK␣ phosphorylation site at p100 C terminus. Thus, the three Cterminal serines of p100 regulate its inducible processing through different mechanisms.
Identification of Additional IKK␣ Phosphorylation Sites within p100 That Regulate p100 Processing-The findings that the C-terminal IKK␣ phosphorylation site (serine 872) of p100 is only partially required for inducible p100 processing suggested the possibility that p100 contains additional phosphorylation sites for IKK␣. To address this possibility, we generated GST fusion proteins containing p100 mutants with progressive C-terminal truncations (Fig. 4A). Indeed, IKK␣ phosphorylated p100 mutants lacking the C-terminal phosphorylation site (p100 1-753 and 1-680) (see Fig. 4B, upper panel,  lanes 1 and 2), thus suggesting the existence of new IKK␣ targets in the N-terminal region of p100. Further truncations from the C terminus revealed that the N-terminal 132 amino acids were sufficient for IKK␣-mediated phosphorylation of the p100 N-terminal region (lane 5); however, a shorter p100 mu-tant containing the first 84 amino acids, p100-(1-84), was no longer phosphorylated by IKK␣ (lane 6). Thus, the major Nterminal phosphorylation sites of p100 appear to be located within a 48-amino acid region covering residues 84 -132.
The N-terminal phosphorylation region of p100 contains four serines, serines 99, 108, 115, and 123. Mutations were introduced to these putative phosphorylation sites either individually or in combination, and the generated GST fusion proteins were subjected to in vitro kinase assays. As shown in Fig. 4C, singular substitution of any of these serines partially affected IKK␣-mediated phosphorylation (upper panel, lanes 2, 3, 4, and  5). However, mutation of all of the four serines simultaneously and largely blocked the phosphorylation by IKK␣ (upper panel,  lane 6). These results suggest that the N-terminal region of p100 contains multiple IKK␣ phosphorylation sites. Consistent with this idea, the N-terminal region of p100 incorporated more radiolabeled phosphates than the C-terminal region of p100 in the in vitro kinase assays (Fig. 4D). Thus, serines 99, 108, 115, and 123 together with serine 872 are the targets of IKK␣.
IKK␣ Phosphorylation Sites within p100 Are Essential for p100 Processing-To investigate the functional significance of the IKK␣ phosphorylation sites within p100 in its inducible processing, we created full-length p100 mutants harboring mutations at these specific residues and examined their inducible processing. Mutation of each of the N-terminal phosphorylation serines resulted in partial reduction in NIK-induced p100 processing (Fig. 5, lanes 2, 4, 6, and 8). When all of the four serines were substituted to alanines, the yielded p100 mutant (p100 4S/A) exhibited a severe but not complete defect in NIK-induced processing (lane 14). Such functional phenotypes were consistent with the phosphorylation patterns of these p100 mutants. The processing deficiency of p100 was enhanced further when both the N-and C-terminal phosphorylation sites were mutated (lane 16). Taken together, these findings strongly suggest that the induction of p100 processing by IKK␣ involves the phosphorylation of p100 at multiple serines located in both the N-and C-terminal regions.
IKK␣ Phosphorylation Sites within p100 Are Involved in Recruitment of ␤-TrCP to p100 and Subsequent p100 Ubiquitination but Not the Recruitment of IKK␣ to p100 -We have reported previously (34) that the inducible processing of p100 involves its physical recruitment of the ubiquitin ligase ␤-TrCP and subsequent ubiquitination. Thus, we examined whether these critical steps of p100 processing require the IKK␣ phosphorylation sites within p100. Co-IP assays were performed to determine the ␤-TrCP binding function of wild type p100 and a p100 mutant harboring mutations at all five I⌲⌲␣-targeting serines (p100 5S/A). As expected, the wild type p100 was bound by ␤-TrCP in cells expressing NIK (Fig. 6A, top panel, lane 2). Further, the p100 mutant lacking its IKK␣ docking site, p100 S866A/S870A, was unable to associate with ␤-TrCP (lane 6). More importantly, the p100 mutant lacking its phosphorylation sites (p100 5S/A) also failed to recruit ␤-TrCP in the presence of NIK (lane 4).
To further confirm the role of p100 phosphorylation sites in its ubiquitination, we directly analyzed the ubiquitin conjugation of wild type and mutant forms of p100 using an in vivo strategy. As we reported previously (15), the wild type p100 became polyubiquitinated in cells transfected with NIK (Fig.  6B, lane 2). In contrast, the p100 mutant lacking its phosphorylation sites was largely inactive in the inducible ubiquitination (lane 4), a phenotype reminiscent of the p100 mutant lacking the IKK␣-docking site (lane 6). Thus, the residues of p100 involved in IKK␣ binding or IKK␣-mediated phosphorylation are essential for recruiting ␤-TrCP and subsequent p100 ubiquitination.  figure). The phosphorylated GST-p100-(754 -900) and its mutants as well as autophosphorylated IKK␣ were indicated (upper panel), and the levels of the wild type and mutant forms of p100 substrates also were monitored by IB analysis of the kinase assay membrane using anti-GST (lower panel). B, NIKinduced processing of p100 involves serine 872 of p100. 293 cells were transfected with the indicated p100 constructs either in the absence (Ϫ) or presence (ϩ) of NIK followed by IB analysis of NIK (lower panel), p100, and p52 processing (upper panel). In the figure, WT, S/A, S/D, SS/DD, and SS/AA stand for p100 wild type, S872A, S872D, S866D/ S870D, and S866A/S870A, respectively. The ratio of p52 to p100 is also indicated.
Because NIK induces p100/IKK␣ association via specific amino acid residues (serines 866 and 870) of p100, it is interesting to test whether the IKK␣ phosphorylation sites within p100 are also involved in NIK-mediated p100/IKK␣ associa-tion. As expected, the p100 mutant harboring serine-to-alanine substitutions at serines 866 and 870 (p100 S866S/S870S) failed to associate with IKK␣ in the presence of NIK (Fig. 6C, top  panel, lane 8). Consistent with its processing deficiency, the FIG. 4. The N terminus of p100 also contains IKK␣-targeting serines. A, schematic representation of GST-p100 fusion mutants. Both Nand C-terminal potential phosphorylation sequences (NPS and CPS), the ␣ helices, the glycine-rich region (GRR), the processing site, and the ARD are also indicated. B, the region between residues 84 and 132 contains IKK␣ phosphorylation site(s). In vitro kinase assays were performed using recombinant IKK␣ and the indicated GST-p100 C-terminal deletion fusion proteins. The phosphorylated GST-p100 C-terminal deletion fusion proteins and autophosphorylated IKK␣ and the levels of those GST fusion proteins were indicated in upper and lower panels, respectively. Note that the ladders in lanes 1-3 were attributed to the partial degradation of GST fusion proteins. C, serines 99, 108, 115, and 123 are targets of IKK␣. GST-p100-(1-132) wild type and its mutants harboring serine substitution(s) were utilized for in vitro kinase assays. Lane 1, GST-p100-(1-132) wild type; lane 2, GST-p100-(1-132) harboring serine 99 substitution with alanine (labeled as S99A); lane 3, GST-p100-(1-132) harboring serine 108 substitution with alanine (labeled as S108A); lane 4, GST-p100 (1-132) harboring serine 115 substitution with alanine (labeled as S115A); lane 5, GST-p100-(1-132) harboring serine 123 substitution with alanine (labeled as S123A); lane 6, GST-p100-(1-132) harboring all of these four serine substitutions with alanine (labeled as 4S/A); Upper panel shows the phosphorylated GST fusion mutants and autophosphorylated IKK␣, whereas the lower panel indicates the levels of those GST fusion proteins. D, the phosphorylation of the N terminus of p100 is stronger than that of its C terminus by IKK␣. Both GST-p100 N-terminal and C-terminal fusion proteins were used for in vitro kinase assays as the same as noted above.
FIG. 5. IKK␣-targeting serines of p100 are required for NIK-mediated p100 processing. p100 wild type and its serine to alanine mutants were transfected alone (Ϫ) or together with NIK (ϩ) followed by analysis of p100 processing using anti-p100N to detect expression of both p100 and p52. The new p100 mutants were designated based on the position of the mutated serine. For example, S99A harbors a substitution of serine 99 by alanine. 4S/A harbors substitutions of serines 99, 108, 115, and 123, whereas 5S/A harbors substitutions of serines 99, 108, 115, 123, and 872. SS/AA is p100 S866A/S870A mutant. The ratio of processing product p52 to the precursor p100 also is indicated in the figure. p100 S866D/S870D mutant also was incapable of IKK␣ association (lane 10). In contrast, the p100 mutants harboring single or multiple mutations at its phosphorylation sites (p100 S872A and p100 5S/A) were fully capable of recruiting IKK␣ in the presence of NIK (lanes 4 and 6). These findings suggest that the recruitment of IKK␣ to p100 and IKK␣-mediated phosphorylation of p100 are two different steps in the induction of p100 processing. DISCUSSION A newly discovered alternative NF-B pathway based on NIKinduced p100 processing via IKK␣ has drawn much attention recently (15)(16)(17). 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). It also has become clear that uncontrolled processing of p100 may contribute to the development of human malignancies, particularly lymphomas (5, 7, 15, 17, 28 -32). Thus, a fundamental understanding of p100 processing will provide important insights into the development of both human immune system and NF-B-associated diseases. Our previous studies using both in vitro biochemical system and in vivo genetically deficient mice clearly demonstrated an essential role for the NIK kinase in regulating p100 processing (15). Further studies indicated that NIK-induced p100 processing specifically involves IKK␣ but not IKK␤ or IKK␥, although the latter two IKK components are critical for canonical NF-B signaling pathway (16,17). Here, we have extended our previous studies by revealing that NIK not only activates IKK␣ but also targets this kinase to its substrate, p100. Once activated and recruited to p100 by NIK, IKK␣ phosphorylates multiple serines located at the N-and C-terminal regions of p100, which triggers the recruitment of ␤-TrCP and subsequent ␤-TrCP-mediated ubiquitination and processing of p100. Interestingly, the IKK␣-docking sites of p100 by NIK are serines 866 and 870, which have been demonstrated to be essential for inducible processing of p100 (15,17). Importantly, neither the kinase activity of IKK␣ nor NIK kinase activity is required for inducible recruitment of IKK␣ to p100, although the kinase activities of both kinases are indispensable for inducible processing of p100. Thus, it is conceivable that the catalytic action of NIK and its IKK␣ docking function are two separate events. However, it seems that the activation of IKK␣ and its association with p100 are still not sufficient to trigger p100 processing, suggesting an additional function and a central role of NIK in p100 processing.
In contrast to constitutive processing of p105, p100 processing is tightly controlled, probably at many levels. Our previous studies indicated that p100 contains at least two negative regulatory domains (15,42). One is dubbed the processinginhibitory domain, which is a region previously identified as a death domain. The other is actually the ankyrin repeat domain (ARD), the characteristic domain of NF-B inhibitors (IBs). Deletion of death domain or ARD will lead to constitutive processing of p100 (15,42). However, it is unlikely that death domain and ARD themselves have specific function in inhibiting p100 processing, because they are very common domains shared by many different proteins. Most notably, the very homologous p105 also has those two domains that shows constitutive processing (1,3). The studies on the structures of NF-B proteins revealed that the N termini of p100 and p105 exhibit different structures (43) that have shown different functions in the processing of p100 and p105. 2 Additionally, it 2 G. Xiao, A. Fong, and S.-C. Sun, unpublished data.
FIG. 6. The recruitment of IKK␣ into p100 and IKK␣-induced phosphorylation of p100 are two sequential steps required for the ␤-TrCP recruitment and subsequent ubiquitylation and processing of p100. A, both the recruitment of IKK␣ into p100 and IKK␣-induced phosphorylation of p100 are essential for NIK-induced binding of p100 to ␤-TrCP. 293 cells were transfected with expression vectors encoding HA-tagged ␤-TrCP and p100 wild type or its IKK␣ recruitment-defective (SS/AA mutant) and phosphorylation-deficient (5S/A) mutants together with either an empty vector (Ϫ) or HA-tagged NIK (ϩ). Cell lysates were subjected to IP using anti-p100N, and the co-precipitated ␤-TrCP was detected by IB using anti-HA-HRP (top panel). The cells lysates were also directly subjected to IB to detect the expression levels of NIK and ␤-TrCP (middle panel) as well as p100 and its processing product p52 (bottom panel) by using anti-HA-HRP and anti-p100N, respectively. p100 5S/A and SS/AA mutants were described in Fig. 5. B, IKK␣-induced phosphorylation of p100 also is essential for polyubiquitination of p100 induced by NIK. 293 cells were transfected with HA-tagged ubiquitin together with the indicated constructs. The p100 wild type and its mutants were isolated by IP with anti-p100N followed by IB using anti-HA-HRP to detect the ubiquitin-conjugated p100 (upper panel). The expression level of ubiquitin was monitored by direct IB using anti-HA-HRP to detect the total ubiquitin-conjugated proteins (lower panel). The expression levels of NIK and p100 were similar to that indicated in A. C, the IKK␣ phosphorylation serines of p100 are dispensable for the recruitment of IKK␣ into p100. The indicated p100 constructs and HA-tagged IKK␣ were transfected alone (Ϫ) or together with NIK (ϩ) followed by co-IP assay using anti-p100N. The co-precipitated IKK␣ was shown in top panel, whereas the expression levels of IKK␣ and NIK as well as p100 and p52 proteins were shown in middle and bottom panels, respectively. The ratio of p52 to p100 also is indicated in this figure. is well demonstrated that the ARD of IBs can associate strongly with the N-terminal Rel homology domain of NF-B proteins (44,45). Taken together, it seems plausible that the death domain of p100 might interact with its N terminus with the help of its ARD, forming a three-dimensional functional domain to control p100 processing. Regarding this hypothesis, we have found that both N and C termini of p100 contain IKK␣ phosphorylation sites (Figs. 3A and 4), which play a critical role in p100 processing (Fig. 5). It is tempting to speculate that the phosphorylation of p100 alters its conformation, which in turns triggers its interaction with ␤-TrCP and subsequent ubiquitination and processing. Clearly, the model needs to be further substantiated by more studies, particularly the structure studies for full-length p100 and p105.
One important function of the interaction between the N and C termini of p100 appears to hide the access of p100 to IKK␣, because IKK␣ fails to associate directly with p100. However, in the presence of NIK, IKK␣ stably binds to p100 (Fig. 1). It seems clear that the IKK␣/p100 interaction is not attributed simply to the result of activation of IKK␣ by NIK, because the activated form of IKK␣ also fails to interact with p100 without NIK, whereas the inactive forms of IKK␣ also can be recruited into p100 in the presence of NIK (Fig. 1). It is possible that the adapter function of NIK may contribute to this interaction. However, it is unlikely that NIK just simply functions as a "bridge" or an "adapter" between IKK␣ and p100, because only low amounts of NIK is required to assemble relatively high amounts of p100/IKK␣ complexes (Fig. 1) and serines 866 and 870 of p100, which are not required for the interaction between p100 and NIK, are essential for the recruitment of IKK␣ to p100 by NIK (Fig. 2), further suggesting that NIK somehow facilitates exposure of those two docking serines of p100, probably through interacting with p100 to modify its conformation. It is also unlikely that the phosphorylation of serines 866 and 870 is the mechanism. Firstly, purified recombinant NIK or IKK fails to phosphorylate these two serines directly (Fig. 3A) (16). Secondly, a kinase-inactive form of NIK also can bring I⌲⌲␣ into p100 complex, although it is not capable of inducing p100 processing (Fig. 1B). Thirdly, p100 S866D/S870D, similar to p100 S866A/S870A mutant, fails to respond to NIKinduced IKK␣ recruitment and p100 processing, whereas p100 S872D has a higher inducible processing ability than wild type p100 (Figs. 3B and 6C). Thus, it is obvious that both the activation and substrate (p100) recruitment of IKK␣ are prerequisites for its role in inducing p100 phosphorylation and processing. However, the activation of IKK␣ and its association with p100 are not sufficient to trigger p100 processing either (Fig. 1B). These data indicated that NIK has an additional function in inducing p100 processing besides activating and recruiting IKK␣ into p100, further suggesting a central role of NIK in this event.
Taken together, our results not only uncover a novel function of NIK but also provide an explanation for why IKK␣ overexpression or its activation by many NF-B inducers fails to induce p100 processing. Although our previous studies have alluded to the fact that NIK is regulated both positively and negatively (33), studies on the mechanisms of NIK regulation should be focused for a complete understanding of how the inducible processing of p100 is regulated by this cellular regulatory pathway.