* This work was supported by a grant from the National Institutes of Health (R01 AI33443) and the Howard Hughes Medical Institute. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
We have examined the role of β-TrCP (β-transducin repeat-containing protein) in the ubiquitination and degradation of IκBβ, one of the two major IκB isoforms in mammalian cells. We demonstrate that β-TrCP interacts specifically with IκBβ, and such interaction is dependent on prior phosphorylation of IκBβ on serines 19 and 23. Interaction with β-TrCP is also necessary for ubiquitination of IκBβ upon stimulation of cells, and deletion of the F-box in β-TrCP abolishes its ability to ubiquitinate IκBβ. Therefore, these results indicate that β-TrCP plays a critical role in the activation of NF-κB by assembling the ubiquitin ligase complex for both phosphorylated IκBα and IκBβ.
IκB kinase complex
β-transducin repeat-containing protein
human immunodeficiency virus
reverse transcriptase polymerase chain reaction
tumor necrosis factor
polyacrylamide gel electrophoresis
The transcription factor NF-κB plays a pivotal role in immune, inflammatory, and stress responses, as well as in early development (
). In nonstimulated cells, NF-κB is retained in an inactive form in the cytoplasm by its interaction with the IκB inhibitory proteins. Mammalian cells contain multiple isoforms of IκB proteins of which IκBα and IκBβ are the best studied (
). Upon stimulation of cells by various cytokines, hormones, or growth factors, a signal-transduction cascade is triggered which leads to the degradation of IκBs and release of NF-κB. The released NF-κB translocates to the nucleus where it up-regulates the transcription of specific target genes (
). The signal-induced degradation of IκB proteins is a critical point in the NF-κB activation pathway. The key step is the phosphorylation of IκB proteins at two specific N-terminal serine residues, which leads to their ubiquitination and subsequent degradation (
). The IKKs phosphorylate IκBα at serines 32 and 36 and mark it for degradation through the ubiquitin-proteosome system. Mutation of either serine residue makes IκBα resistant to phosphorylation and degradation (reviewed in Ref.
During ubiquitin-dependent degradation, ubiquitin molecules activated by ubiquitin-activating enzyme E1 are attached to specific lysine residues on the target protein by a ubiquitin-conjugating enzyme (E2), together with a ubiquitin ligase (E3) that is specific for the substrate (
). Recently an F-box/WD40 protein called β-TrCP (β-transducin repeat-containing protein) was shown to be the substrate-recognition component of the ubiquitin ligase responsible for phosphorylation-dependent ubiquitination of IκBα (
). β-TrCP recognizes IκBα phosphorylated at Ser-32 and Ser-36 through its WD40 domain, whereas the F-box motif recruits additional proteins including Skp1 and Cullin to form the Skp1-cullin-F-box (SCF) ubiquitin ligase complex (
). β-TrCP belongs to a growing family of proteins containing F-boxes that are involved in assembling the SCF complex. Aside from the F-box, these proteins have another protein-protein interaction module in their C terminus, namely a WD or leucine-rich repeat (LRR) domain. These C-terminal domains mediate the interaction of SCF complexes with their substrates and determine specificity of substrate recognition. β-TrCP has been implicated in the ubiquitination of CD4 (through HIV protein Vpu) (
). All these proteins share similar N-terminal inducible phosphorylation sites with the consensus sequence of DSGψXS (ψ represents a hydrophobic residue and X represents any amino acid.). Therefore, the inducible phosphorylation of these N-terminal serine residues is the critical step that allows recruitment of β-TrCP and subsequent ubiquitination of these proteins (
). Instead, one study has reported that serines 19 and 23 of IκBβ are constitutively phosphorylated in unstimulated cells, suggesting that the regulation of IκBβ might differ more fundamentally from that of IκBα (
). Because phosphorylation of IκBβ by the IKKs does not induce a mobility shift in SDS-PAGE, andin vivo labeling experiments have been inconclusive, signal-induced phosphorylation of IκBβ remains to be unequivocally demonstrated.
). The underlying mechanism responsible for these differences is unclear although one possible explanation for the slower kinetics of IκBβ degradation might be lower efficiency of ubiquitination of phosphorylated IκBβ. Understanding the details of the pathway by which phosphorylated IκBβ is ubiquitinated and degraded is therefore important for fully decoding the differential regulation of IκBα and IκBβ. The identification of β-TrCP as the recognition element of IκBα ubiquitin-ligase provides an opportunity to directly test whether IκBβ also undergoes signal-induced phosphorylation and degradation, and whether it is mediated through β-TrCP.
We report in this manuscript that β-TrCP specifically interacts with IκBβ in stimulated cells. This interaction requires serines 19 and 23 because mutation of these residues completely abolishes this interaction. We also demonstrate that phosphorylation-induced ubiquitination of IκBβ requires the F-box of β-TrCP, suggesting that both IκBα and IκBβ appear to be ubiquitinated and degraded through the same pathway. Therefore the differential regulation of IκBα and IκBβ is most likely because of differences in other steps in the activation pathway of NF-κB.
Cell Cultures, Antibodies, and Reagents
293, HeLa, and COS cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. The anti-flu mouse monoclonal antibody (12CA5) was produced and purified in this laboratory. Anti-flag monoclonal antibody M5 and anti-flag M2 affinity gel were purchased from Sigma. All other antibodies were purchased from Santa Cruz Biotechnology. Protein A-Sepharose was purchased from Amersham Pharmacia Biotech.
Cloning of Human β-TrCP and β-TrCPΔF
Total RNA was isolated from HeLa cells using TRIzol™ Reagent (Life Technologies, Inc.) and used for RT-PCR to amplify β-TrCP cDNA with appropriate 5′- and 3′-primers. A FLAG-epitope coding sequence was inserted after the starting codon. The 1.7-kilobase PCR product was subcloned intoBamHI and XbaI sites of expression vector pCDNA3 (Invitrogen) and sequenced.
To delete the F-box from β-TrCP, two internal primers were designed to flank the boundary sequences outside of F-box region and used individually with the 5′- or 3′-primers described above to amplify the N terminus or C terminus of β-TrCP. The 500-base pair N-terminal and 1.1-kilobase C-terminal PCR products were then used as templates in the sequential PCR with 5′- and 3′-primers. The resulting β-TrCPΔF was subcloned into BamHI-XbaI sites of pCDNA3, and its sequence was confirmed by DNA sequencing.
Subconfluent 293 cells were transfected with 500 ng of pBIIX luciferase reporter construct, along with different amounts of β-TrCP or β-TrCPΔF constructs. The total transfected DNA amounts were equalized with empty pCDNA3 vector. After 36 h, cells were treated with or without 20 ng/ml TNF-α for 4 h before harvest for luciferase assay (Promega).
Transfection, Immunoprecipitation, and Immunoblotting
Cells were grown in 10 centimeter plates to 40% confluence and transfected with indicated DNA using FuGENE™ 6 (Roche Molecular Biochemicals). After incubation for 36 h, cells are treated with or without TNF-α (10 ng/ml) for 30 min before being lysed with TNT lysis buffer (200 mm Tris-HCl, pH 8.0, 200 mm NaCl, 1% Triton-100) supplemented with protease inhibitors. In immunoprecipitation experiments, cell lysates were incubated with 20 μl of anti-flag M2 affinity gel for 3 h or 10 μl of anti-IκBβ (C-20) along with 20 μl of protein A-Sepharose for 4 h at 4 °C. Immobilized immuno-complexes were washed with TNT three times, boiled in SDS loading buffer, and resolved on 10% SDS-PAGE. Proteins were transferred to Immobilon transfer membrane (Millipore Corp.) and blotted with indicated primary antibody for 3 h at room temperature and appropriate secondary antibody for 1 h. Immunoreactive bands were visualized by ECL.
RESULTS AND DISCUSSION
β-TrCP Is Involved in NF-κB Activation and β-TrCPΔF Acts As a Dominant Negative Regulator of NF-κB Activation
Human β-TrCP was cloned using RT-PCR from HeLa cells with primers designed according to the published protein sequence. An F-box deletion mutant, β-TrCPΔF, was generated by deleting the F-box region from leucine 148 to leucine 192 (Fig. 1) (
). Both wide type and ΔF mutant of β-TrCP were flag-tagged at their N terminus, cloned into the expression vector pcDNA3, and their integrity verified by DNA sequencing and in vitroexpression.
We first investigated the role of β-TrCP in NF-κB activation. We transfected 293 cells with β-TrCP or β-TrCPΔF construct, together with a luciferase reporter gene pBIIX-luc, which harbored two NF-κB binding sites in its promoter region. As shown previously (
), introduction of β-TrCPΔF into the cells significantly inhibited NF-κB activation in a concentration-dependent manner (data not shown). The F-box motif has been found to be important for associating with Skp1, which in turn binds to Cullin and an E2 enzyme to form a functional ubiquitin-conjugating complex. Therefore the β-TrCPΔF construct would bind to phosphorylated IκB but fail to recruit the other components, thus inhibiting the degradation of IκB and activation of NF-κB. Surprisingly, we also found that transfection of wild type β-TrCP also inhibited NF-κB activation, although to a lesser extent than the F-box deletion mutant (data not shown). The explanation for this observation is unclear, but one possibility is that overexpression of β-TrCP results in the accelerated degradation of some other component that is required for activation and nuclear translocation of NF-κB.
β-TrCP Binds to Phosphorylated IκBβ
To examine whether β-TrCP directly interacts with phosphorylated IκBβ, we conducted immunoprecipitation experiments in transfected cells. IκBα and IκBβ were transfected into 293 and HeLa cells respectively, along with either FLAG-tagged wild type β-TrCP or FLAG-tagged TrCPΔF construct. Cells were incubated with the proteosome inhibitor, calpain inhibitor 1, before treatment with TNF-α. Cell lysates were precipitated with anti-flag affinity gel, and the immobilized immuno-complex was immunoblotted for IκBα or IκBβ. The experiment confirmed that interaction between IκBα and β-TrCP is only observed in TNFα-stimulated cells (Fig.2A). Similarly, IκBβ failed to associate with β-TrCP in unstimulated cells, but bound efficiently to β-TrCP in TNF-α stimulated cells (Fig.2B). In both instances, deletion of the F-box deletion did not affect the ability of β-TrCP to interact with IκBα or IκBβ (Fig. 2, A and B). This result is therefore consistent with the notion that β-TrCP interacts with its phosphorylated substrate through its WD domain, and this binding is independent of the F-box.
Treatment with TNF-α has been shown to cause the phosphorylation of IκBα at the N-terminal serine residues 32 and 36 (
). To ascertain whether β-TrCP binds only to IκBβ phosphorylated at serines 19 and 23, we used an IκBβ mutant, IκBβ19/23 SS/AA, in which serines 19 and 23 were replaced with nonphosphorylatable alanines. As shown in Fig.2C, IκBβ19/23 SS/AA mutant failed to associate with β-TrCP even upon TNF-α treatment. This confirmed that the interaction between IκBβ and β-TrCP is contingent upon prior phosphorylation of IκBβ at the N terminus. Interestingly, an IκBβ19/23 SS/DD mutant in which the two serines were substituted by aspartic acid, to mimic the phosphorylated state, also failed to bind to β-TrCP or β-TrCPΔF. This experiment demonstrates the stringent substrate specificity of β-TrCP for phosphate groups in the IκB proteins. Similar specificity of interaction had been observed in earlier studies where it was demonstrated that only a phosphopeptide encompassing the IκBα degradation motif, but not a serine-to-glutamate-substituted peptide, could compete with intact IκBα for ubiquitin conjugation (
β-TrCP Promotes Ubiquitination of Phosphorylated IκBβ in Vivo
To directly assess whether β-TrCP is a component of the ubiquitin ligase for IκBβ, we transfected COS cells with wild type or ΔF mutant β-TrCP along with HA(flu)-tagged IKKβ. COS cells appear to lack certain components in the signaling pathways leading to NF-κB activation and hence do not respond to traditional inducers of NF-κB.
Therefore to help bypass this difficulty, we co-transfected HA(flu)-tagged IKKβ, FLAG-tagged wild type or ΔF mutant β-TrCP, and IκBβ. The IκBβ bound to β-TrCP was analyzed by immunoprecipitation of β-TrCP with anti-FLAG antibody, followed by immunoblotting with IκBβ antisera. Under these conditions, where transfection of IKKβ presumably led to continuous phosphorylation of IκB proteins, multiple IκBβ bands with increasing molecular weights were detected in β-TrCP transfected cells. (Fig.3A, upper panel,lane 4). In contrast, only a single band corresponding to IκBβ is observed in cells transfected with the dominant negative β-TrCPΔF construct (Fig. 3A, upper panel,lane 6). Although we could detect forms of IκBβ containing one or two ubiquitin molecules using the IκBβ antibody, we did not observe polyubiquitinated forms under these experimental conditions. Because the levels of polyubiquitinated IκB forms are very low, probably because they are rapidly degraded, we repeated the experiment and exposed the ECL blot for significantly longer periods. Under these conditions, we could detect low amounts of higher molecular weight forms of IκBβ that probably represent polyubiquitinated forms of the protein (Fig. 3B). To further characterize the higher molecular weight IκBβ immunoreactive bands, we immunoblotted the anti-flag-immunoprecipitated complexes with ubiquitin antibody. In contrast to the immunoblot with the IκBβ antibody, the slower migrating bands were readily detected with the ubiquitin antibody (Fig.3A, middle panel, lane 4). The explanation for why the ubiquitin antibody detects the higher molecular weight species more readily is probably because of the far greater number of epitopes that are presented by the polyubiquitinated forms. As expected, deletion of the F-box in β-TrCP (β-TrCPΔF) almost completely blocked the formation of ubiquitin-IκBβ conjugates (Fig.3A, middle panel, lane 6). Therefore in cells transfected with IKKβ, β-TrCP is directly involved in IκBβ ubiquitination.
To further confirm that β-TrCP promotes the ubiquitination of IκBβ, whereas the β-TrCPΔF suppresses it, we examined the state of IκBβ in β-TrCP and β-TrCPΔF transfected cells by directly immunoprecipitating IκBβ itself. Transfection of either IκBβ or IκBβ along with β-TrCP does not lead to significant ubiquitination of IκBβ (Fig. 3C, upper panel,lanes 1 and 2). However, upon activation by IKKβ transfection, IκBβ is polyubiquitinated (Fig. 3C,upper panel lane 3). Transfection of wild type β-TrCP significantly increased the level of ubiquitination of IκBβ (Fig.3C, lane 4), whereas cells transfected with β-TrCPΔF failed to generate ubiquitinated forms of IκBβ (Fig.3C, lane 5). The ubiquitination of IκBβ is dependent on serines 19 and 23 because a mutant IκBβ containing alanines in these positions could not be ubiquitinated (Fig.3C, lane 6). Therefore these observations are in agreement with earlier results examining the binding of mutant IκBβ with β-TrCP (Fig. 2, B and C).
In summary, we report that β-TrCP binds specifically to the inducibly phosphorylated IκBβ and promotes its ubiquitination. Our findings further help establish the role of β-TrCP as a component of the ubiquitin ligase for IκB proteins, and demonstrate that the signal-induced phosphorylation of IκBβ by IKKs is a critical step that precedes their ubiquitination and degradation. Therefore, differences in the regulation of IκBα and IκBβ must be because of differences in other steps in the pathway. For example, it is possible that IκBβ complexes contain an additional regulatory component that determines the rate of degradation of ubiquitinated IκBβ proteins, thus explaining their slower rate of degradation. Alternatively, such an associated regulatory protein may influence the ability of IκBβ to be efficiently phosphorylated by IKK. Finding the answers to these questions remains a challenge for the future.
We thank Dr. Dola Sengupta for comments on the manuscript.