Phosphorylation of the PEST Domain of IκBβ Regulates the Function of NF-κB/IκBβ Complexes*

Activation of transcription factor NF-κB involves the signal-dependent degradation of basally phosphorylated inhibitors such as IκBα and IκBβ. The gene encoding IκBα is under NF-κB control, which provides a negative feedback loop to terminate the induced NF-κB response. However, recent studies have identified a hypophosphorylated pool of IκBβ that shields nuclear NF-κB from inhibition by newly synthesized IκBα. In the present work, we provide three lines of evidence indicating that this protection mechanism is regulated by the C-terminal PEST domain of IκBβ. First, disruption of two basal phosphoacceptors present in the IκBβ PEST domain (Ser-313 and Ser-315) yields a mutant that forms ternary complexes with NF-κB and its target DNA-binding site. Second, based on in vitromixing experiments, these ternary complexes are resistant to the inhibitory action of IκBα. Third, mutants of IκBβ that are defective for phosphorylation at Ser-313 and Ser-315 fail to efficiently block NF-κB-directed transcription in vivo, whereas replacement of these two IκBβ residues with a phosphoserine mimetic generates a fully functional repressor. Taken together, our findings suggest that the functional fate of NF-κB when bound to IκBβ is critically dependent on the phosphorylation status of the IκBβ PEST domain.

Inducible members of the NF-B/Rel family of transcription factors mediate rapid cellular responses to stress-related, mitogenic, and pro-apoptotic signals (reviewed in Ref. 1). The most well characterized form of NF-B consists of two DNAbinding subunits, termed p50 and RelA, which are sequestered in the cytoplasm of unstimulated cells by basally phosphorylated IB proteins (1). Prior studies have defined a general three-step process for NF-B activation involving signal-de-pendent phosphorylation and ubiquitination of IB at N-terminal sites, which targets the inhibitor for degradation by the 26S proteasome (1). In turn, this proteolytic event exposes subcellular localization motifs on NF-B that direct the transcription factor to the nuclear compartment (1).
Within a given cell type, another layer of complexity in the NF-B signaling pathway arises from the expression of multiple isoforms of IB, including IB␣ and IB␤ (1). Although both of these inhibitors are basally phosphorylated and subject to signal-dependent degradation, recent studies indicate that IB␣ and IB␤ are regulated by distinct mechanisms that affect the duration of nuclear NF-B activity (1)(2)(3). Specifically, activation cues that selectively trigger IB␣ degradation are typically associated with a transient pattern of nuclear NF-B expression, whereas the concomitant loss of IB␤ correlates with a persistent NF-B response (2,3). In further contrast to IB␤, the gene encoding IB␣ is rapidly induced by NF-B (1). This relationship establishes a negative feedback pathway in which newly synthesized IB␣ can facilitate a transient mode of NF-B action (4). However, under stimulatory conditions that lead to prolonged NF-B activation, cells may express a hypophosphorylated form of IB␤ that binds NF-B and shields it from feedback inhibition by IB␣ (5,6). The precise molecular mechanism by which NF-B/IB␤ complexes escape from the inhibitory action of IB␣ during a persistent NF-B response remains unclear.
In this report, we provide evidence for the presence of regulatory serine residues within the C-terminal PEST domain of IB␤ that affect its capacity to either terminate or preserve the functional expression of nuclear NF-B. Specifically, when overexpressed in mammalian cells, mutants of IB␤ containing alanine substitutions for Ser-313 and Ser-315 fail to efficiently block NF-B-directed transcription relative to the wild type inhibitor. These PEST mutants of IB␤ are hypophosphorylated in vivo and form ternary complexes with NF-B and DNA that are resistant to dissociation by IB␣ in vitro. In contrast, replacement of Ser-313 and Ser-315 with aspartic acid, which can act as a phosphoserine mimetic (7,8), rescues the NF-B inhibitory function of IB␤. These findings indicate that the PEST domain of IB␤ plays dual roles in the control of NF-B activity, depending on the phosphorylation status of Ser-313 and Ser-315.
Functional and Biochemical Assays-CAT assays were performed as described (14) using a liquid scintillation counting method (19). To measure NF-B DNA binding activity, nuclear extracts were prepared (20) and incubated with a 32 P-labeled B-pd probe using published reaction conditions (21). Nucleoprotein complexes were resolved on native 5% polyacrylamide gels and visualized by autoradiography (21). Protein-DNA cross-linking experiments (14) were conducted with a photoreactive derivative of the B-pd probe containing 5-bromo-2Ј-deoxyuridine 5Ј-triphosphate (22). DNA-protein adducts were fractionated by immunoprecipitation with either RelA-specific antiserum or monoclonal anti-FLAG antibodies and resolved by SDS-PAGE.

The PEST Domain of IB␤ Is Basally Phosphorylated at
Ser-313 and Ser-315-Primary structural (2, 11) and functional (3,14,23) analyses have established that IB␤ is a tripartite inhibitor containing (i) an N-terminal response domain required for signal-dependent breakdown, (ii) a central NF-B-interactive domain composed of six ankyrin repeat motifs, and (iii) a C-terminal PEST domain that harbors two consensus phosphorylation sites for casein kinase II (CKII). We recently demonstrated that the PEST domain of IB␤ is basally phosphorylated at serine residues (14). As shown in Fig. 1A, five serines in this domain are clustered within or near the two consensus CKII motifs ((Ser/Thr)-Xaa-Xaa-(Glu/Asp); Ref. 24).
To initially explore which of these PEST residues are phosphoacceptors, a series of specific amino acid substitutions were introduced into the full-length IB␤ protein (Fig. 1A), and the corresponding cDNA expression vectors were transfected into A293T cells. After metabolic radiolabeling with 32 P i , ectopic IB␤ proteins were immunopurified and analyzed by SDS-PAGE (Fig. 1B). Substitution of alanine for Ser-313 and Ser-315 (Mu-1) resulted in a 3-fold reduction in phosphoryl group transfer to wild type IB␤ (Fig. 1B, lanes 1 and 2), whereas disruption of all five serines in this PEST region almost completely abolished basal phosphorylation (Mu-3; Fig. 1B, lane 4). In contrast, replacement of Ser-313 and Ser-315 with the phosphoserine mimetic aspartic acid (7,8) partially restored the phosphorylation defect of Mu-1 (ϳ75% of wild type; Fig. 1B,  lane 3). However, these aspartic acid mutations failed to rescue phosphorylation when introduced into the serine-depleted PEST background (Mu-4; Fig. 1B, lane 5). All of these PEST mutants were expressed at comparable levels in vivo (data not shown). These data suggest that the IB␤ PEST domain is phosphorylated on multiple serines by a cooperative mechanism that is dependent on phosphoryl group transfer to Ser-313 and/or Ser-315.
To directly test whether IB␤ is phosphorylated at Ser-313 and Ser-315, site-directed mutants containing threonine rather than serine at both positions were prepared and overexpressed in A293T cells. After biosynthetic radiolabeling with 32 P i , ectopic IB␤ proteins were immunopurified and subjected to twodimensional phosphoamino acid analysis. Consistent with our prior findings (14), control experiments performed with wild type IB␤ revealed phosphorylation exclusively at serine residues (Fig. 1C). In contrast, analysis of the threonine mutant (designated S313T/S315T) revealed the presence of both phosphoserine and phosphothreonine. Similar qualitative results were obtained with IB␤ mutants harboring single threonine substitutions at either position (data not shown). These findings indicate that Ser-313 and Ser-315 both serve as phosphoacceptors in vivo.
Phosphorylation  (Fig. 1), we next explored their potential to recapitulate the unusual functional phenotype of the hypophosphorylated form of IB␤ described by Suyang et al. (5).
For these studies, A293T cells were cotransfected with expression vectors for the p50 and RelA subunits of NF-B along with plasmids encoding FLAG epitope-tagged IB␤ proteins. Nuclear extracts were prepared and cross-linked to 32 P-labeled B oligonucleotides. To specifically detect NF-B/IB␤ complexes bound to target DNA, the resultant adducts were immunoprecipitated with anti-FLAG antibodies and analyzed by SDS-PAGE for the presence of RelA ( Fig. 2A). PEST mutants containing alanine substitutions for Ser-313 and Ser-315 formed stable ternary complexes with NF-B and DNA, as evidenced by co-immunoprecipitation with RelA (Mu-1; Fig. 2A,  lane 3). Similar results were obtained with the serine-depleted PEST mutant of IB␤ (Mu-3; Fig. 2A, lane 5). In contrast, PEST mutants containing phosphoserine mimetics at positions 313 and 315 associated with DNA-bound NF-B at much lower Transfected cells were radiolabeled with 32 P i , and ectopic IB␤ was purified from whole cell lysates by immunoprecipitation with anti-FLAG antibodies. Immunoprecipitates were resolved by SDS-PAGE, transferred to a polyvinylidine difluoride membrane, and subjected to autoradiography. Molecular sizes are given in kilodaltons. C, A293T cells (1 ϫ 10 6 ) were transfected with expression vectors (7.5 g) for either wild type IB␤ (WT) or the IB␤ mutant S313T/S315T. After 36 h of culture, transfectants were metabolically labeled with 32 P i (4 h), and ectopic IB␤ proteins were immunopurified using anti-FLAG antibodies. Immunoprecipitates were subjected to phosphoamino acid analysis in parallel with standards. The positions of phosphorylated (P-) amino acids and the sample origin (Ori) are indicated. efficiencies, provided that the neighboring serine residues were preserved ( Fig. 2A, lanes 4 and 6). We conclude that phosphorylation of Ser-313 and Ser-315 in IB␤ is necessary but not sufficient to block ternary complex formation. Coupled with the finding that the phosphorylation status of Ser-313 and Ser-315 affects phosphoryl group transfer to vicinal serines (Fig. 1B), it seems likely that the hypophosphorylated form of endogenous IB␤ described by Suyang et al. (5) lacks modifications at these two PEST sites.
PEST Mutants of IB␤ Protect NF-B from Inhibition by IB␣-Another proposed property of the hypophosphorylated form of IB␤ expressed in vivo is its ability to shield NF-B from inhibition by newly synthesized IB␣ (5,6). To test whether this protection mechanism is regulated specifically by PEST-directed phosphorylation events, lysates were prepared from A293T cells after cotransfection with expression vectors for p50 and RelA. In parallel, we prepared lysates enriched for either wild type IB␣ or a phosphorylation-defective mutant of IB␤ that formed ternary complexes with NF-B and DNA (Mu-3; Figs. 1A and 2A). Extracts containing NF-B were incubated with a photoreactive B-pd probe in the presence of Mu-3, and the resultant nucleoprotein complexes were crosslinked and immunoprecipitated with RelA-specific antiserum. As shown in Fig. 2B, radiolabeled RelA-DNA adducts were readily detected in IB␣-deficient reactions (Fig. 2B, lane 1, top  panel). The addition of IB␣ to these reaction mixtures failed to disrupt the DNA binding function of NF-B when associated with Mu-3 (Fig. 2B, lanes 2-5, top panel). In contrast, IB␣ potently inhibited NF-B DNA binding activity in control reaction mixtures lacking Mu-3 (Fig. 2B, lanes 2-5, bottom  panel), which confirmed the integrity of IB␣ preparations used in these studies. As such, ternary complexes containing this phosphorylation-defective PEST mutant of IB␤ are resistant to dissociation by IB␣, a property consistent with that proposed for hypophosphorylated IB␤ in vivo (5,6).
Regulation of NF-B-directed Transcription by IB␤ PEST Mutants-In addition to its capacity to bind DNA, the RelA subunit of NF-B contains a C-terminal transactivation domain (1). Although the data shown in Fig. 2A demonstrate that complexes containing NF-B and phosphorylation-defective forms of IB␤ bind target DNA in vitro, the in vivo activity of NF-B in the presence of these PEST mutants remained untested. To address this critical issue, A293T cells were cotransfected with a CAT reporter plasmid driven by two B enhancer elements along with expression vectors for p50, RelA, and IB␤. Under our overexpression conditions, NF-B stimulated reporter gene activity ϳ35-fold over basal levels in control transfectants lacking ectopic IB. As shown in Fig. 3A, wild type IB␤ completely blocked the transactivation function of NF-B. Similar results were obtained with a PEST mutant harboring phosphoserine mimetics in place of Ser-313/Ser-315 (Mu-2). In contrast, phosphorylation-defective mutants of IB␤ that formed ternary complexes with NF-B and DNA in vitro failed to efficiently block NF-B-dependent transcription in vivo. All of these functional results correlated strongly with the pattern of NF-B DNA binding activity observed in gel shift experiments that were conducted with nuclear extracts from the same transfectants (Fig. 3B). Taken together, these in vivo data suggest that the functional fate of NF-B when bound to FIG. 2. Phosphorylation-defective PEST mutants of IB␤ form ternary complexes with NF-B and DNA that are resistant to dissociation by IB␣. A, A293T cells (1 ϫ 10 6 ) were cotransfected with pCMV4 expression vectors for RelA (2 g), p50 (2 g), and the indicated FLAG-tagged derivatives of IB␤ (1 g). After 36 h of growth, nuclear extracts were prepared from recipient cells, added to reaction mixtures containing a photoreactive 32 P-labeled B probe, and crosslinked by exposure to UV light (300 nm for 30 min). Photoreactive adducts were immunoprecipitated with anti-FLAG antibodies, resolved by SDS-PAGE, and visualized by autoradiography. Molecular mass standards are given in kilodaltons. WT, wild type. B, whole cell extracts were prepared from A293T cells transfected with expression vectors for RelA and p50 (3.75 g each), IB␣ (7.5 g), or IB␤ mutant Mu-3 (7.5 g). Extracts enriched for NF-B (2.5 g) were added to reaction mixtures containing a photoreactive 32 P-labeled B probe in the presence (upper panel) or the absence (lower panel) of IB␤ mutant Mu-3 (5 g). Following a 10-min incubation, the indicated DNA-binding mixtures were supplemented with increasing doses (0.25, 0.5, 1.0, and 2.5 g) of whole cell extracts derived from IB␣-expressing transfectants (lanes 2-5). Products were cross-linked with UV light (300 nm, 30 min) and subjected to immunoprecipitation with RelA-specific antiserum. DNAprotein adducts were resolved by SDS-PAGE and visualized by autoradiography. IB␤ is critically dependent on the phosphorylation status of the IB␤ PEST domain. DISCUSSION Recent studies have established that the PEST domain of IB␤ is basally phosphorylated on serines in vivo (14). However, the precise role of this domain in the regulation of NF-B activity remained unclear. One clue to its function emerged from experiments showing that bacterially expressed forms of IB␤ lacking these basal modifications associate with NF-B but fail to prevent DNA binding (5). Consistent with this, phosphatase treatment of purified IB␤ from mammalian cells results in the loss of NF-B inhibitory activity (25). Furthermore, specific serine mutations within the PEST domain of IB␤ have been identified that disrupt its ability to associate efficiently with c-Rel, another member of the NF-B family of transcription factors (14).
The present report provides strong evidence that the PEST domain of IB␤ controls whether NF-B/IB␤ complexes are either latent or competent for transcription. We have found that serines clustered within the IB␤ PEST domain are phosphorylated via a cooperative mechanism involving requisite modifications at Ser-313 and Ser-315. In keeping with the behavior of wild type IB␤, mutants defective for phosphorylation at these two residues associate stably with NF-B. In sharp contrast to the wild type inhibitor, these PEST mutants fail not only to prevent NF-B DNA binding in vitro but also to terminate NF-B-directed transcription in vivo. This functional defect is completely rescued by replacing the disrupted PEST sites with a phosphoserine mimetic. We conclude that basal phosphorylation of Ser-313 and Ser-315 of IB␤ is required for the formation of latent NF-B/IB␤ complexes. However, phosphoryl group transfer to additional serines within this region of the PEST domain (Fig. 1A) is necessary to generate the fully functional inhibitor.
Unlike IB␤, IB␣ is encoded by an NF-B-responsive gene (1). This interplay normally provides a feedback mechanism to ensure the transient action of NF-B. In this regard, Suyang et al. (5) recently identified a hypophosphorylated form of IB␤ in untransfected lymphocytes and proposed that this species contributes to the chronic activation of NF-B under certain stimulatory conditions. In support of their proposal, in vitro mixing experiments with bacterially expressed proteins showed that recombinant IB␤ associates efficiently with NF-B and shields it from neutralization by IB␣ (5). However, these prior studies did not define the sequences in endogenous IB␤ that mediate this protective function.
In the present work, we identified phosphorylation-defective PEST mutants with properties that closely mimic this unusual functional phenotype. As determined with IB proteins from mammalian cells, mutants defective for phosphorylation at Ser-313 and Ser-315 form stable ternary complexes with NF-B and target DNA that are resistant to dissociation by IB␣ in vitro. These results highlight a potential regulatory role for the IB␤ PEST domain in the repression of NF-Bdirected transcription by IB␣. Further resolution of this issue awaits insights into the phosphorylation status of the PEST domain of IB␤ during a transient versus persistent NF-B response.