Research ArticleBiochemistry

The NF-κB subunit RelB controls p100 processing by competing with the kinases NIK and IKK1 for binding to p100

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Sci. Signal.  27 Sep 2016:
Vol. 9, Issue 447, pp. ra96
DOI: 10.1126/scisignal.aad9413

Intermolecular competition for activation or inhibition

Noncanonical nuclear factor κB (NF-κB) signaling requires cleavage of the RelB-bound p100 precursor to generate the transcriptionally active p52:RelB heterodimer in a process dependent on the kinases NIK and IKK1. However, p100:RelB dimers also form kappaBsomes, multiprotein complexes that sequester NF-κB subunits to inhibit gene expression. Fusco et al. found that the function of p100 was determined by how RelB was bound to a transitional complex consisting of p100, NIK, and IKK1. If the binding of RelB to p100 displaced NIK and IKK1 from p100, p100 was not phosphorylated or cleaved, and instead, kappaBsomes formed. On the other hand, failure of RelB to displace the kinase complex before p100 phosphorylation resulted in the formation of the active p52:RelB dimer.

Abstract

The heterodimer formed by the nuclear factor κB (NF-κB) subunits p52 and RelB is the product of noncanonical signaling in which the key event is the proteolytic processing of p100 to generate p52. The kinases NF-κB–inducing kinase (NIK) and inhibitor of κB kinase 1 (IKK1; also known as IKKα) are activated during noncanonical signaling and play essential roles in p100 processing. In resting cells, RelB remains associated with unprocessed p100 as a transcriptionally inert p100:RelB complex, which is part of a larger assembly with other NF-κB factors known as the “kappaBsome.” We investigated how these two different RelB-containing complexes with opposing effects on target gene transcription are formed. We found that RelB controls the extent of both p100 processing and kappaBsome formation during noncanonical signaling. Within an apparently “transitional” complex that contains RelB, NIK, IKK1, and p100, RelB and the NIK:IKK1 complex competed with each other for binding to a region of p100. A fraction of p100 in the transitional complex was refractory to processing, which resulted in the formation of the kappaBsome. However, another fraction of p100 protein underwent NIK:IKK1-mediated phosphorylation and processing while remaining bound to RelB, thus forming the p52:RelB heterodimer. Our results suggest that changes in the relative concentrations of RelB, NIK:IKK1, and p100 during noncanonical signaling modulate this transitional complex and are critical for maintaining the fine balance between the processing and protection of p100.

INTRODUCTION

RelB, a member of the nuclear factor κB (NF-κB) family of transcription factors, is involved in the regulation of diverse physiological activities, including development, survival, immunity, and inflammation (1, 2). RelB confers transcriptional activity by associating primarily with two other NF-κB family members, p50 and p52 (35). RelB also associates with RelA, but the resulting RelA:RelB heterodimer is incapable of binding to DNA (6). Controlled processing of the p100 precursor protein to generate the NF-κB p52 subunit and the subsequent formation of the p52:RelB heterodimer are hallmarks of the noncanonical NF-κB signaling pathway. Two protein kinases, NF-κB–inducing kinase (NIK) and inhibitor of κB (IκB) kinase 1 (IKK1; also known as IKKα), cooperate to control p100 processing. NIK is thought to activate the kinase activity of IKK1 by phosphorylating its activation loop residues Ser176 and Ser180 (1, 711). However, the precise mechanisms underlying p100 processing and the formation of the p52:RelB heterodimer remain unclear.

Both p105, the precursor of the NF-κB p50 subunit, and p100 function as inhibitors of diverse NF-κB proteins, including NF-κB dimers that contain the processed p50 or p52 subunits (1214). The inhibitory activity of the precursors results from their formation into heterogeneous high–molecular mass protein complexes known as “kappaBsomes” (15). Combinatorial assembly of four p100 molecules and four NF-κB subunits (RelA, c-Rel, RelB, and p52) form the p100-centric kappaBsomes (16). However, p105 and p100 are distinct in their inhibition specificity; p105 interacts with RelA and c-Rel, but not with RelB. In addition to the p105- and p100-specific kappaBsomes, p105 and p100 can also associate with each other. Thus, kappaBsomes are heterogeneous, each inhibiting a different pool of NF-κB subunits.

The ability of p100 to inhibit NF-κB activity is essential for bone development, B cell proliferation, and the control of infection (1720). How both the inhibitory and transcriptional activities of p100 (through its processing into p52) are properly balanced is not known. It is presumed that noncanonical signaling leads simply to degradation of the C-terminal NF-κB inhibitory domain of p100 within the p100:RelB complex, thereby resulting in the formation of p52:RelB heterodimers. This assumption was challenged by a report that showed that processing required newly synthesized p100 molecules. In that study, the new and old p100 molecules were distinguished from each other through differential stable isotope labeling by amino acids in cell culture, and the fate of p100 was monitored by mass spectrometry (21). The regulation of NF-κB by IκB in the noncanonical pathway therefore contrasts substantially with that in canonical signaling, where preformed NF-κB dimers bind to prototypical IκB-type inhibitors and are activated through complete proteolysis of the inhibitors.

Because RelB associates with p100 and the processing of p100 into p52 leads to the generation of p52:RelB heterodimers, a major transcriptionally active NF-κB dimer during noncanonical signaling, we hypothesized that RelB might play a critical role in the processing of p100 to p52. Here, we found that RelB controlled both p100 processing and the formation of the p100-containing kappaBsomes, two activities with opposing effects on the NF-κB transcriptional regulatory system. We observed that the NIK:IKK1 complex removes most p100 from the cytosol either through processing or complete degradation during noncanonical signaling in the absence of RelB. In the presence of RelB, NIK:IKK1 complex and RelB compete for binding to p100. Furthermore, this competition occurs within a complex that contains RelB, NIK, IKK1, and p100. The processing of p100 takes place within this transient multiprotein complex, which results in p52:RelB heterodimer formation, whereas the displacement of NIK:IKK1 from the multiprotein complex before p100 processing led to the formation of kappaBsomes.

RESULTS

RelB plays a key role in balancing the relative amounts of p100 and p52

RelB is unique among NF-κB subunits because of its domain organization. Although, like other NF-κB subunits, it contains the Rel homology region (RHR), this segment in RelB is more proteolytically sensitive than that of other subunits, which suggests that RelB has a lower folding stability and likely contributes to its specificity for p50 or p52 subunits when forming heterodimers in vivo. RelB also contains a unique segment at its N terminus, which is referred to as the leucine zipper domain. We previously reported that RelB and p100 bind to one another through a complex interface that involves interactions between multiple domains on both subunits (5). We reasoned that a more complicated mode of interaction between RelB and p100 could play a role in regulating the stability and processing of p100. We first tested this hypothesis by examining p100 abundance in wild-type (WT) and Relb−/− mouse embryonic fibroblasts (MEFs). In the absence of RelB, the steady-state abundance of p100 was reduced compared to that in WT cells (Fig. 1A). The abundance of p100 was further reduced after prolonged stimulation of the Relb−/− cells with an anti–lymphotoxin β receptor (LTβR) antibody (α-LTβR), an agonist of the noncanonical NF-κB signaling pathway. Stimulus-responsive processing of p100 remained intact in Relb−/− cells because the amount of p52 detected was increased over the time of stimulation, which suggests that RelB might play a role in the stabilization of p100 during noncanonical signaling. To further test whether RelB played a role in p100 stabilization, we reconstituted Relb−/− cells with WT RelB and then stimulated the cells with α-LTβR. We found that p100 protein abundance was substantially stabilized in the reconstituted cells (Fig. 1A). Under similar conditions, the stabilities of the p105 and p50 proteins were not markedly affected (fig. S1A).

Fig. 1 RelB inhibits p100 degradation.

(A) Top: WT MEFs, Relb−/− MEFs, and Relb−/− MEFs reconstituted with WT relb [relb WT transgenic (Tg)] were stimulated with α-LTβR for the indicated times. Samples were then analyzed by Western blotting [immunoblotting (IB)] with antibodies against the indicated proteins to monitor the processing of p100 to generate p52. β-Actin was used as a loading control. Western blots are representative of three experiments. Bottom: Densitometric analysis of the ratio of p52 abundance to p100 abundance for the indicated samples. Data are means ± SEM of three independent experiments. (B) Top: WT MEFs were left untreated, whereas Relb−/− MEFs were treated with 10 μM MG132 for the indicated times before the samples were analyzed by Western blotting with an antibody against p100. β-Actin served as a loading control. Bottom: Densitometric analysis of the intensity of the p100 band, normalized to that of β-actin, for each sample. Data are means ± SEM of three experiments. (C) RelB blocks p100 processing in human embryonic kidney (HEK) 293T cells. Top: HEK 293T cells were transiently transfected with the indicated combinations of plasmids encoding Flag-p100, green fluorescent protein (GFP)–RelB, and hemagglutinin (HA)–NIK. Forty-eight hours later, the cells were lysed and analyzed by Western blotting with antibodies against the indicated targets. β-Actin served as a loading control. Western blots are representative of three experiments. Bottom: Densitometric analysis of the ratio of p52 abundance to p100 abundance for the indicated samples. Data are means ± SEM of three independent experiments. (D) RelA plays no role in p100 degradation. Top: HEK 293T cells were transiently transfected with the indicated combinations of plasmids encoding Flag-RelA, GFP-RelB, Flag-p100, and HA-NIK. Forty-eight hours later, the cells were lysed and analyzed by Western blotting with antibodies against the indicated targets. β-Actin served as a loading control. Western blots are representative of four experiments. Bottom: Densitometric analysis of the ratio of p52 abundance to p100 abundance for the indicated samples. Data are means ± SEM of four independent experiments.

We next investigated the mechanism of homeostatic p100 degradation in Relb−/− cells by examining the effect of MG132, a small-molecule inhibitor of the proteasome. MG132 resulted in a modest stabilization of p100 in Relb−/− MEFs, suggesting a role for the proteasome in the degradation of p100 in the absence of RelB (Fig. 1B). We also compared p100-encoding mRNA abundance in WT and Relb−/− MEFs in response to α-LTβR. We found that RelB was partly involved in the activation of p100 transcription in response to α-LTβR (fig. S1B). These observations suggest that RelB enhances p100 protein abundance in two different ways: as a transcriptional activator of p100 expression and as a stabilizer of p100 protein.

To further investigate how RelB affected p100 in cells, we monitored the cellular distribution and dynamic state of p100 by imaging it in the presence and absence of RelB. We generated a p100 construct that was tagged with GFP at the N terminus and mCherry at the C terminus, and the synthetic gene encoding the GFP-p100-mCherry fusion protein was introduced into two different MEF cells: p100−/− and p100−/−Relb−/− cells. Confocal microscopy revealed that green and red fluorescence were merged in both unstimulated and α-LTβR–stimulated p100−/− MEFs expressing the p100 fusion protein (fig. S1C, top). We observed that both p100 and p52 were mobilized to the nucleus in stimulated cells. Western blot analysis confirmed that the processing of the p100 fusion protein was similar to that of endogenous p100 in WT MEFs (fig. S1D). However, the green and red fluorescence were observed in distinct regions of the cytoplasm, and the two colors were not merged when the p100 fusion protein was expressed in the absence of RelB in p100−/−Relb−/− cells (fig. S1C, middle). When the same cells were stimulated with α-LTβR, we observed increased amounts of GFP-p52 in the nucleus; however, little mobilization of p100 into the nucleus was observed. Western blot analysis revealed the disappearance of p100 in p100−/−Relb−/− cells 6 hours after stimulation with α-LTβR (fig. S1D). Finally, we tested the status of p100 and RelB when coexpressed in p100−/−Relb−/− MEFs. Under these conditions, the GFP-p100-mCherry fusion protein behaved similarly to that in p100−/− MEFs, suggesting that the heterologous expression system effectively mimics the native state of the system (fig. S1C, bottom). Overall, these results suggest that RelB stabilizes p100 in both unstimulated and stimulated cells.

The effect of RelB on p100 protein stabilization was further evaluated through transfection-based experiments. It was previously shown that exogenous p100 undergoes processing in the presence of constitutively active NIK, bypassing the requirement for upstream signals (7). Under conditions of constant amounts of active NIK, we examined p100 processing in the presence of increasing amounts of RelB. We found that p100 processing was progressively inhibited as RelB concentrations increased (Fig. 1C). In contrast, overexpression of RelA had no effect on p100 processing in these cells (Fig. 1D). Our results suggest that RelB is antagonistic to the generation of the p52:RelB heterodimer. However, formation of the p52:RelB heterodimer is a key consequence of noncanonical signaling. A dynamic balance mediated by RelB and NIK enables both the processing and stabilization of p100 during noncanonical signaling.

RelB competes with NIK for binding to p100

While we performed the experiments described previously, we observed a curious relationship between NIK, p100, and RelB: the abundance of NIK protein decreased as that of RelB increased (Fig. 1C). In contrast, increasing the cellular concentration of RelA had no such effect on NIK abundance (Fig.1D). To test whether this effect was a result of an interaction between RelB and NIK, we performed coimmunoprecipitation experiments with transfected HEK 293T cells. We found that NIK and p100 were coimmunoprecipitated as a stable complex only in the absence of RelB (Fig. 2A). Increasing the concentration of RelB in the cells resulted in the decreased association of NIK with p100 (Fig. 2A). To lend further support to this observation, we added cell extracts containing increasing amounts of RelB to the p100:NIK complex bound to agarose beads and tested for the release of NIK from the complex. We found that RelB, but not RelA, resulted in the displacement of NIK from the p100:NIK complex (Fig. 2B). We further tested this relationship between NIK and RelB in MEFs by investigating whether the abundance of NIK was stabilized under resting or stimulated conditions in Relb−/− cells relative to that in WT or Relb−/− cells reconstituted with RelB. There were substantially increased amounts of NIK protein in both unstimulated and α-LTβR–stimulated Relb−/− cells than in WT cells or RelB-reconstituted Relb−/− cells (Fig. 2C). We found that there was no difference in NIK mRNA abundance in any of these cells, which was consistent with a role for the posttranscriptional regulation of NIK (fig. S2A). Together, our results suggest that RelB inhibits the processing of p100 by actively displacing NIK from p100 such that free NIK undergoes degradation.

Fig. 2 RelB competes with NIK and IKK1 for binding to p100.

(A) Top: HEK 293T cells transiently transfected to express the indicated combinations of constructs were subjected to immunoprecipitation (IP) of Flag-p100 with anti-Flag M2 agarose beads. Samples were then analyzed by Western blotting with antibodies against the indicated targets. Western blots are representative of three experiments. Bottom: Densitometric analysis of the ratio of p52 abundance to p100 abundance for the indicated samples. Data are means ± SEM of three independent experiments. (B) Top: HEK 293T cells were transiently transfected with plasmids encoding Flag-p100 and HA-NIK and then subjected to immunoprecipitation with anti-Flag M2 agarose beads. Another set of HEK 293T cells was transfected with two different amounts of plasmids encoding GFP-RelB or GFP-RelA, and lysates of these cells were incubated with beads bound to the Flag-p100:NIK complex for 30 min. Cell lysates (Input) and immunoprecipitated samples were analyzed by Western blotting with antibodies against the indicated proteins. Western blots are representative of three experiments. Bottom: Densitometric analysis of the abundance of NIK normalized to that of p100 for the indicated samples. Data are means ± SEM of three independent experiments. (C) WT MEFs, Relb−/− MEFs, and Relb−/− MEFs reconstituted with WT relb were treated with α-LTβR for the indicated times. Cell lysates were analyzed by Western blotting to assess NIK abundance. β-Actin served as a loading control. Western blots are representative of three experiments. (D) HEK 293T cells were transfected with the indicated combinations of plasmids encoding full-length HA-tagged NIK, an HA-tagged N-terminal truncated NIK NIKΔN324, Flag-p100, and GFP-RelB. Forty-eight hours later, the cells were lysed, and cell lysates were analyzed by Western blotting with antibodies against the indicated targets. Western blots are representative of three experiments. (E) HEK 293T cells were left untreated or were treated with the indicated concentrations of the IKK inhibitor XII before being transfected with plasmids encoding Flag-p100 and HA-NIK. Twenty-four hours later, the cells were analyzed by Western blotting with antibodies against the indicated proteins. β-Actin served as a loading control. Western blots are representative of three experiments. (F) RelB blocks the IKK1-stimulated processing of p100 in HEK 293T cells. HEK 293T cells were transiently transfected with the indicated combination of plasmids encoding Flag-p100, HA-IKK1, and GFP-RelB. Forty-eight hours later, the cells were lysed and analyzed by Western blotting with antibodies against the indicated proteins. β-Actin served as a loading control. Western blots are representative of three experiments. (G) A model depicting competition between the NIK:IKK1 complex and RelB for binding to p100 and its processing. The NIK:IKK1 complex induces the processing of p100, whereas RelB displaces the NIK:IKK1 complex that is bound to p100 to prevent its processing. The model also depicts the reduction in the abundance of free NIK (p100-unbound) by IKK1.

How does p100 control NIK stability? NIK is maintained at low amounts in resting cells by two different mechanisms. The first of these involves tumor necrosis factor (TNF) receptor–associated factor 2 (TRAF2) and TRAF3. TRAF3 interacts with the N-terminal domain of NIK, leading to the continuous degradation of NIK, which also requires TRAF2 and cellular inhibitor of apoptosis 1 and 2 (cIAP1/cIAP2) (2224). An N-terminally truncated NIK construct (ΔN324) escapes degradation mediated by TRAFs, cIAPs, and the proteasome and, in turn, processes p100 into p52 constitutively. Such an NIK mutant is responsible for the oncogenic transformation of cells (25). In the second mechanism, NIK activity is reduced by IKK1, which stimulates NIK degradation (26, 27). To test whether either or both of these pathways was functional in our system, we transfected HEK 293T cells with a plasmid encoding NIKΔN324 and found that this mutant was also unstable in the absence of p100 but was stabilized in the presence of p100 (Fig. 2D). This finding suggests that the TRAF pathway does not play a role in the degradation of NIK.

To test the IKK1 activity, we used the IKK inhibitor XII (Calbiochem), a small-molecule IKK inhibitor that is equally potent toward IKK1 and IKK2 (28). We found that the inhibitor compound served to stabilize NIK protein in HEK 293T cells (Fig. 2E). When used at 50 μM, the IKK inhibitor XII prevented IKK activation, as measured by blockade of the phosphorylation of the IKK activation loop (fig. S2B). Because IKK2 is not activated by NIK (1), we concluded that IKK1 activity was blocked by this inhibitor. Pretreatment of HEK 293 cells with the same inhibitor before the cells were transfected with plasmids encoding NIK and p100 resulted in reduced processing of p100 (Fig. 2E). The α-LTβR–induced processing of p100 was also inhibited in MEFs pretreated with the inhibitor (fig. S2C). These results suggest that, similar to the activity of endogenous NIK during noncanonical signaling, overexpressed NIK can activate endogenous IKK1.

We further tested whether, similar to NIK, IKK1 also competed with RelB for binding to p100. We found that overexpression of IKK1 resulted in the increased processing of exogenous p100 (Fig. 2F). RelB inhibited the processing of p100 by competing with IKK1 for binding to p100, similar to its competition with NIK (Fig. 2F). Together, these data suggested the mechanism of interplay between NIK, p100, RelB, and IKK1 (Fig. 2G). First, NIK and IKK1 function in conjunction with each other, even under conditions of overexpression, to induce p100 processing. Second, both NIK and IKK1, likely as a complex, compete with RelB for binding to p100. Third, active IKK1 blocks the accumulation of NIK by inducing its degradation, possibly through the phosphorylation of three C-terminal serines of NIK, as was shown previously (26). Last, p100 prevents the IKK1-dependent degradation of NIK by binding to both kinases.

RelB favors kappaBsome formation

We previously reported that p105 and p100 remain as a large complex (kappaBsome) bound to NF-κB subunits in unstimulated cells (15). We further showed that p100 associates with all NF-κB subunits, including RelB, to form large molecular assemblies (16). RelB does not bind to p105, and it requires p100 for its stability. These results and our current finding regarding competition between the NIK:IKK1 complex and RelB for binding to p100 led us to hypothesize that RelB might play a specific role in p100-centric kappaBsome formation by preventing NIK accumulation during noncanonical signaling. We examined the nature and integrity of the kappaBsomes in the presence and absence of RelB under steady-state conditions and upon stimulation. We prepared cytoplasmic extracts from both WT and Relb−/− MEFs before stimulation and 6 or 12 hours after stimulation with α-LTβR. Extracts were fractionated by passing them through a Superose 6 size-exclusion column and then were subjected to Western blot analysis (fig. S3A). The overall amounts of p100 were reduced in all fractions of Relb−/− MEF extracts compared to those in the extracts of WT cells (fig. S3A). This difference was even more substantial in the extracts of stimulated cells. Both p100 and p52 were nearly absent in the fractions of Relb−/− MEFs 12 hours after stimulation with α-LTβR. In contrast, fractions of WT cell extracts showed the persistent presence of p100 in the high–molecular mass fractions (maximal abundance in fraction #24) before and after stimulation with α-LTβR (fig. S3A).

We further investigated kappaBsome formation in transfected cells. We fractionated the cytoplasmic extracts of HEK 293T cells that were transfected with plasmids encoding p100, RelB, RelA, and c-Rel in different combinations. The elution profile of p100 in the presence or absence of different combinations of NF-κB enabled us to estimate the roles of these proteins in the molecular assembly between p100 and the NF-κB proteins. These studies revealed that the presence of RelB was essential for high–molecular mass complex formation because RelB, on its own, associated with p100 to form a high–molecular mass complex (Fig. 3A). Although RelA and c-Rel interacted with p100 in the absence of RelB, they did not form larger assemblies, suggesting that only RelB mediated the formation of native kappaBsomes (Fig. 3B). We noted that the elution profile of p100 in Relb−/− cell extracts was only marginally different from that in WT cell extracts (fig. S3A), whereas the difference in peak volumes in transfected cell extracts with and without RelB was substantially larger (Fig. 3B). This is most likely due to the association of p100 with other endogenous partner proteins in the absence of RelB. Together, these results provide insight into the relationship between p100 and RelB in that they assemble into stable high molecular weight complexes.

Fig. 3 RelB favors p100-kappaBsome formation.

(A) Top: HEK 293T cells were transfected with the indicated combinations of plasmids encoding Flag-p100, GFP-RelB, HA-RelA, and Myc-cRel. Forty-eight hours later, cell extracts were fractionated by size-exclusion chromatography (Superose 6), and the fractions were analyzed by Western blotting (IB) with an antibody against p100. Boxes denote the fractions with maximal p100 elution. Western blots are representative of three experiments. Bottom: Densitometric analysis of the abundance of p100 for the indicated samples. Data are representative of three independent experiments. (B) Extracts of HEK 293T cells transfected with the indicated combinations of plasmids encoding Flag-p100, HA-RelA, and Myc-cRel were fractionated as described in (A) and analyzed by Western blotting with antibodies against the indicated proteins. Rectangles denote the fractions with maximal amounts of p100. (C) WT MEFs were left unstimulated, were stimulated with α-LTβR for 12 hours alone, or were washed after the stimulation and incubated for a further 12 hours. Cell extracts were then subjected to immunoprecipitation with anti-p100 antibody and Western blot (IB) analysis with antibodies against the indicated proteins. Western blots are representative of three experiments. (D) A model depicting how RelB protects p100 from processing during stimulation. This model is a continuation of the model shown in Fig. 2G. The p100:RelB complex further associates with other NF-κB subunits to form the kappaBsome. Together, the models shown here and in Fig. 2G depict the opposing events that occur during noncanonical signaling.

We next tested whether RelB remained associated with p100 during cellular stimulation and after the stimulus was withdrawn. WT MEFs were treated with α-LTβR for 12 hours or were cultured for another 12 hours after withdrawal of the initial 12-hour stimulus. We observed that both RelA and RelB remained bound to p100 both 12 hours after the cells were stimulated with α-LTβR and 12 hours after the stimulus was withdrawn (Fig. 3C). It is reasonable to assume that in the absence of RelB, NF-κB might be constitutively active because RelB favors kappaBsome formation, which enables p100 to act as an inhibitor of NF-κB, including RelA and cRel. We found that the abundance of IκBα in Relb−/− MEFs was increased compared to that in WT cells, which suggests that the lack of p100-kappaBsome is compensated for by the increased abundance of IκBα (fig. S3B). As expected, the abundance of IκBα in p100−/− MEFs was also increased compared to that in WT cells. We also found that the TNF-α–stimulated degradation of IκBα remained intact in all cell types. These data suggest that the primary role of RelB in kappaBsome formation is to displace the NIK:IKK1 complex to protect the pool of p100 from undergoing processing (Fig. 3D). The p100:RelB complex then further associates with other NF-κB subunits to form mature kappaBsomes.

RelB protects p100 from complete degradation

The substantial reduction in p100 abundance upon prolonged stimulation in the absence of RelB shown earlier (Fig. 1A) could result from complete degradation of p100, rapid processing and degradation of the p52 subunit, or both. We wanted to investigate how p100 became mostly undetectable after prolonged stimulation of Relb−/− cells with α-LTβR. We transfected HEK 293T cells to coexpress p100 and NIK with or without RelB, added cycloheximide (CHX) to the cells for different time periods, and then monitored p100 degradation and processing by Western blot analysis. We found that overexpressed p100 was processed in the presence of NIK (Fig. 4A). Under this condition, p100 was completely undetectable within 3 hours after CHX treatment in the absence of RelB but not in its presence. These results suggest that p100 undergoes both complete degradation and processing in stimulated cells and that these processes are partly inhibited by RelB.

Fig. 4 RelB protects p100 from complete degradation.

(A) HEK 293T cells were transiently transfected with the indicated combination of plasmids encoding Flag-p100, HA-tagged NIK-WT or NIK-inactive mutant, and GFP-RelB. Forty-eight hours later, the cells were left untreated or were treated with CHX for the indicated times before cell extracts were analyzed by Western blotting with antibodies against the indicated targets. Western blots are representative of three experiments. (B) Relb−/− MEFs reconstituted with either WT RelB or the RelB Y300A mutant were left unstimulated or were stimulated with α-LTβR for the indicated times. The processing of p100 was monitored by Western blot analysis with antibodies against the indicated proteins. β-Actin served as a loading control. Western blots are representative of three experiments.

We next investigated whether RelB remained associated with p100 while p100 was being processed. We generated a mutant RelB (RelB Y300A) that is unable to form a heterodimer with p52 (4). When Relb−/− MEFs expressing this dimerization-defective mutant were stimulated with α-LTβR, the extent of p100 processing was reduced compared to that in Relb−/− MEFs expressing WT RelB and, after prolonged stimulation, both p100 and RelB were nearly undetectable and little or no p52 was present (Fig. 4B). The RelB Y300A mutant still bound to p100 but not to p52 (fig. S4, A and B). We previously reported that RelB and p100 interact through multiple sites. One of these interactions occurs between the two dimerization domains and another occurs between the activation domain of RelB and a region in p100 located near the processing site of p100. Complete degradation of p100 in the presence of the RelB Y300A mutant suggests that the lack of formation of stable p52:RelB heterodimers led to the complete degradation of p100. This also suggests that RelB, if present, imposes itself onto p100 and that processing and degradation of p100 occurs when RelB remains bound to p100.

Thus, we generated a reciprocal mutation in p100 (Y247A) located within the p52 dimerization domain that prevents p52 from forming heterodimers with RelB and other NF-κB subunits (4). When p100−/− MEFs were reconstituted with the p100 Y247A mutant, they exhibited an even more severe phenotype than that of WT, p100-expressing cells, such that no p52 was observed before or after the cells were stimulated with α-LTβR (fig. S4C). The p100 Y247A mutant protein still bound to RelB (fig. S4D) but was unable to interact with the RHR of p52 (fig. S4E). This suggests that stable dimer formation between p100 and RelB is an important prerequisite for the processing of p100. The inability of p52 to dimerize with any NF-κB subunit because of the alteration of a critical dimer-forming residue in p100 prevented the processing of p100. MEFs expressing the RelB Y300A mutant showed basal p100 processing because p52 still could form dimers with other NF-κB subunits. Together, these observations suggest that complete degradation and processing follow a similar path and that the improper structural integrity of the components within the kappaBsomes alters the mode from processing (partial degradation) to complete degradation.

A transitional p100:RelB:NIK:IKK1 complex precedes both the processing of p100 and kappaBsome formation

So far, we showed that physical interactions between RelB and p100 are essential for the stability of p100. The stabilization of p100 results from the ability of RelB to displace an NIK:IKK1 complex and hence block the proteasomal degradation of p100. We further showed that the formation of p52:RelB heterodimers and the kappaBsome apparently proceed through a common intermediate. Because p52:RelB heterodimer formation requires the action of the NIK:IKK1 complex, we hypothesized that the common intermediate might also contain the NIK:IKK1 complex.

To test for the presence of our predicted multiprotein complex, we immunoprecipitated RelB or p100 from the extracts of transfected HEK 293T cells overexpressing RelB, NIK, and p100 and analyzed the precipitated samples for bound components by Western blotting (fig. S5A). If p100 formed complexes only with RelB or NIK:IKK1, then immunoprecipitation with an anti-RelB antibody should not result in the detection of NIK. However, we found that RelB was capable of coimmunoprecipitating NIK together with p100 (fig. S5A). To test whether these molecules also interacted in untransfected cells, we performed coimmunoprecipitation experiments with untreated and α-LTβR–treated MEFs. The results of these experiments also suggested that there was a three-way association among RelB, NIK, and p100 (Fig. 5A). In this system, the abundance of NIK was expected to be very low. Therefore, we coincubated the cells with MG132 and α-LTβR for an hour, immediately before cell lysis was performed. We also repeated the coimmunoprecipitation experiments in cells that were not treated with MG132 and found that p100 interacted with RelB, NIK, and IKK1 in response to stimulation with α-LTβR (Fig. 5B). These results suggest that competition between NIK:IKK1 and RelB occurs within a complex in which all of the competing molecules are present.

Fig. 5 p100, NIK:IKK1, and RelB form a transitional complex.

(A) WT MEFs were left untreated or were stimulated with α-LTβR for 8 hours. The cells were also treated with 10 μM MG132 for 1 hour before they were lysed and subjected to immunoprecipitation with antibodies against p100, NIK, or RelB or with immunoglobulin G (IgG) as a control. Lysates (Input) and immunoprecipitated samples were analyzed by Western blotting (IB) with antibodies against the indicated proteins. Western blots are representative of three experiments. (B) WT, Relb−/−, and p100−/− MEFs were left untreated or were stimulated with α-LTβR for 8 hours. Cell lysates were subjected to immunoprecipitation with antibody against p100, and lysates (Input) and immunoprecipitated samples were analyzed by Western blotting (IB) with antibodies against the indicated proteins. Western blots are representative of three experiments. (C) A model showing the generation of either p52 or kappaBsomes from p100 in a single transient complex. In the transient complex, p100, NIK:IKK1, and RelB remain associated in a competing manner. If RelB causes the displacement of the NIK:IKK1 complex before p100 is phosphorylated by these kinases, then kappaBsomes will be formed. On the other hand, if p100 is phosphorylated while RelB remains bound to p100 (perhaps in a different conformation), this will result in the generation of the p52:RelB heterodimer. This model is an extension of the models shown in Figs. 2G and 3D.

To further test the association between p100, RelB, and NIK, we cotransfected HEK 293T cells with plasmids encoding p100 and NIK or with plasmids encoding p100, RelB, and NIK and fractionated cell extracts by Superose 6 size-exclusion column. The fractions were collected and analyzed by Western blotting with a phospho-specific antibody against pSer866 of p100, which is a marker for p100 processing (fig. S5B). Whereas maximal phosphorylated p100 abundance was observed in fraction #23 in the absence of RelB, it shifted to fractions #21 and #22 in the presence of RelB, indicating a heavier complex containing RelB. If p100 processing or degradation had occurred from the p100:NIK complex, then the presence of RelB would not have affected the elution profile of the p100:NIK complex (indicated by phosphorylated p100). This finding suggests that the NIK:IKK1 complex stimulates the processing of p100 by associating with the p100:RelB complex.

In pancreatic cancer cells, the abundances of both NIK and RelB are increased compared to those in pancreatic ductal epithelial cells (2931). This is somewhat inconsistent with our observations that increased amounts of RelB should prevent NIK from binding to p100 and should prevent constitutive degradation of NIK. To investigate how NIK escapes regulation by p100, we tested the associations among NIK, RelB, and p100 in MIA PaCa-2 pancreatic cancer cells (fig. S5C). We found that in these cells, the association of RelB p100 occurred to a lesser extent than did their association in HeLa cells, whereas the NIK-p100 association was similar to that in HeLa cells. It was previously shown that the increased abundance of NIK in HeLa cells is due to the deregulation of the TRAF pathway (31). Together, these results suggest that both NIK and RelB are disconnected from RelB-p100-NIK circuitry in pancreatic cancer cells.

DISCUSSION

Our study unravels two highly intertwined, but opposing, activities of p100 and RelB in noncanonical NF-κB signaling, leading us to propose a model for p52:RelB heterodimer generation by the NIK:IKK1 complex (Fig. 5C). Both RelB and NIK:IKK can associate with p100 to form multiprotein complexes, but their effects on p100 are opposing. Whereas the intrinsic activity of RelB is to stimulate formation of the kappaBsome by making multisite contacts with p100, the intrinsic activity of the NIK:IKK1 complex is to phosphorylate p100. Thus, RelB primarily acts as an inhibitor of p100 processing. However, RelB must enable some processing of p100 to p52. This challenging dual tasking of RelB is solved elegantly: RelB uses its unique ability to form the p100-centric kappaBsome within which other NF-κB subunits may interact with p100 (3234). Our study suggests that the generation of p52 occurs within the context of such a multiprotein complex.

The ability of RelB to dimerize with the N-terminal domain of p52 also ensures the formation of the p52:RelB heterodimer, which is the critical, transcriptionally active NF-κB dimer of noncanonical signaling. The generation of both p52:RelB and p100:RelB from a single intermediate serves to maintain a delicate balance in the amounts of both complexes (Fig. 5C). Thus, the competition between RelB and NIK:IKK1 for binding to p100 is an active process, and the relative concentrations of NIK:IKK1 and RelB determine the final outcome. In addition, p100 appears to undergo complete degradation, which might also be influenced by the nature of the intermediate complex. Our work provides insights into how noncanonical signaling is transduced through balancing the amounts of distinct complexes with opposing activities. Increased or reduced processing of p100 causes cancer and defects in immunity (3539). In all these cases, aberrant processing could be due to the defect in competition between RelB and the NIK:IKK1 complex for p100 binding as shown here. Although our study provides an initial mechanistic model of p100 processing, many questions remain unanswered. For example, the requirement of a steady-state amount of kappaBsome and its fate upon stimulation are not clear nor is the distinct time lag between cellular stimulation and the processing of p100 to generate p52. Furthermore, beyond complex association and p100 phosphorylation, the roles of IKK1 and NIK in processing are also unknown. A temporal dissection of the levels and activities of different complexes and their physical interplay is required for a better understanding of noncanonical NF-κB signaling.

MATERIALS AND METHODS

Antibodies and reagents

Primary antibodies against RelB (sc-226), RelA (sc-372), cRel, β-actin (sc-1615), and IKK1 and all of the horseradish peroxidase–conjugated secondary antibodies were purchased from Santa Cruz Biotechnology. Antibodies against Flag (M2), GFP (A6-455), and HA were obtained from Sigma, Invitrogen, and Covance, respectively. Antibodies against NIK (#4994), phosphorylated IKK1/IKK2 (Ser180/Ser176), and phosphorylated p100 (Ser866/870; #4810) were from Cell Signaling. Antibodies against p100 and p52 were gifts from BioBharati LifeScience (BBL, India) and N. Rice (National Cancer Institute, Frederick, MD), respectively. MG132 and CHX were purchased from Sigma. The IKK inhibitor XII (also referenced under CAS 928655-63-4) was purchased from Calbiochem. Protein G and A/G agarose beads were gifts from BBL, India.

Generation of stable cell lines

All constructs were cloned into the pBABE vector, and stable cell lines were prepared as described previously (18).

Mammalian cell culture and transient protein expression

WT and knockout MEFs were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% calf serum, 2 mM glutamine, and antibiotics. HEK 293T and HeLa cells were maintained in DMEM supplemented with 10% fetal bovine serum, 2 mM glutamine, and antibiotics. MIA PaCa-2 cells were maintained in DMEM supplemented with 10% fetal bovine serum, 2% horse serum, 2 mM glutamine, and antibiotics. HEK 293T were transfected with polyethylenimine (Polysciences; cat #23966-2). Cell lysates were prepared 24 to 48 hours after transfection or as indicated in the figure legends.

Cytoplasmic and nuclear fractionation and size-exclusion chromatography

Cytoplasmic and nuclear fractionation of cells and Superose 6 size-exclusion chromatography were performed as described previously (15).

Western blotting and immunoprecipitations

Cell extracts were prepared by harvesting cells and lysing them with a buffer containing 20 mM tris-HCl (pH 8.0), 0.2 M NaCl, 1% Triton X-100, 2 mM Na2VO4, 2 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride, 5 mM 4-nitrophenyl phosphate di(tris) salt, and protease inhibitor mixture (Sigma). Protein (15 to 30 μg) from total cell lysates or nuclear and cytoplasmic extracts was resolved by SDS–polyacrylamide gel electrophoresis followed by transfer to a nitrocellulose membrane. Western blot analysis was then performed with specific antibodies. For coimmunoprecipitation reactions, cells were lysed in lysis buffer [20 mM tris-HCl (pH 7.5), 200 mM KCl, 5 mM MgCl2, 10 mM Na2VO4, 1 mM DTT] containing 0.5% Triton X-100, 0.5% NP-40, and 1× protease inhibitor cocktail (Sigma) for 30 min at 4°C. The lysates were clarified by centrifugation at 16,000g for 10 min and were incubated with either prebound Protein G Sepharose bead (GE Healthcare or BBL, India) overnight at 4°C or anti-Flag M2 affinity gel (Sigma; A2220) for 1 hour at 4°C. Subsequently, the beads were washed thrice with lysis buffer containing 0.5% Triton X-100, and the bound proteins were analyzed by Western blotting as described earlier.

In vitro interaction assay

For in vitro interaction assays (as depicted in Fig. 2B), HEK 293T cells were transfected with plasmids expressing Flag-p100 and HA-NIK. Forty-eight hours later, the cells were lysed with lysis buffer [20 mM tris-HCl (pH 7.5), 200 mM KCl, 5 mM MgCl2, 10 mM Na2VO4, 1 mM DTT] containing 0.5% Triton X-100, 0.5% NP-40, and 1× protease inhibitor cocktail (Sigma) and then immunoprecipitated with anti-Flag M2 agarose beads (Sigma). Another set of HEK 293T cells was transfected with increasing amounts of plasmids encoding either RelB or RelA. Forty-eight hours later, the cells were lysed, and equal amounts of cell lysates containing different amounts of RelB or RelA were incubated with Flag-p100–bound beads at 4°C for 30 min. The reaction was stopped with 2× Laemmli dye, and different protein amounts were analyzed by Western blotting.

RNA isolation and reverse transcription PCR

RNA was isolated from MEFs using TRIzol reagent (Invitrogen) according to the manufacturer’s protocol. Reverse transcription polymerase chain reaction (PCR) was performed with a Reverse Transcription Kit (BBL, India). The following primers were used: p100: forward, 5′-GGTCTGCACCCACCTGCAGC-3′; reverse, 5′-AGGGCGACCTGGAAGTCC-3′; MAP3K14: forward, 5′-CGAGGGGGTCCTGCTTACTG-3′; reverse, 5′-CGAGCTCAGACCAGCACAGGCC-3′; and GAPDH: forward, 5′-TTCACCACCATGGAGAAGGC-3′; reverse, 5′-TGTCATGGATGACCTTGGCC-3′.

SUPPLEMENTARY MATERIALS

www.sciencesignaling.org/cgi/content/full/9/447/ra96/DC1

Fig. S1. Aberrant subcellular distributions of p52 and p100 in Relb−/− cells.

Fig. S2. NIK and RelB compete for binding to p100.

Fig. S3. RelB stimulates kappaBsome formation.

Fig. S4. RelB protects p100 from complete degradation.

Fig. S5. RelB, NIK:IKK1, and p100 form a multiprotein complex.

REFERENCES AND NOTES

Acknowledgments: We thank A. Hoffmann for the WT and knockout MEF cell lines and for the use of a fluorescence microscope. We thank M. Karin for the MIA PaCa-2 pancreatic cancer cell line. We thank M. Sen, M. C. Mullero, T. Huxford, A. Hoffmann, and T. Biswas for critically reading the manuscript. We also thank S. Basak for the discussion. Funding: A.J.F. was supported by a Heme and a Cell Growth and Regulation Training Grant for predoctoral research and by a predoctoral fellowship from the University-wide AIDS Research Program. V.Y.-F.W. was supported by the Science and Technology Development Fund, Macao S.A.R. (FDCT) project 023/2016/A1. Z.T. was supported by a postdoctoral fellowship from the Growth Regulation and Oncogenesis Training Grant. This work is supported by funding from the NIH grants AI064326 and PO1 GM071862. Author contributions: A.J.F., A.M., V.Y.-F.W., and Z.T. designed and performed the experiments; A.J.F., A.M., V.Y.-F.W., and G.G. analyzed the data; C.W. provided the reagents; and G.G. wrote the manuscript with input from A.M. and A.J.F. Competing interests: The authors declare that they have no competing interests.
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