Role of Monoubiquitylation on the Control of IκBα Degradation and NF-κB Activity

The NF-κB pathway is regulated by multiple post-translational modifications including phosphorylation, ubiquitylation and SUMOylation. Many of these modifications act on the natural inhibitor IκBα modulating its capacity to control signal-mediated NF-κB activity. While the canonical pathway involving the phosphorylation and polyubiquitylation of IκBα has been well characterized, the role of these post-translational modifications in the control of basal NF-κB activity has not been deeply explored. Using the recently developed Tandem-repeated Ubiquitin Binding Entities (also known as ubiquitin traps) to capture ubiquitylated proteins, we identified monoubiquitylated forms of IκBα from multiple rat organs and cell types. The identification of these forms was demonstrated through different procedures such as immunoprecipitations with specific ubiquitin antibodies or His6-Ubiquitin pull downs. Monoubiquitylated forms of IκBα are resistant to TNFα-mediated degradation and can be captured using TUBEs, even after proteasome inhibitors treatment. As it occurs for monoSUMOylation, monoubiquitylation is not dependent of the phosphorylation of IκBα on the serines 32/36 and is not optimally degraded after TNFα stimulation. A ubiquitin-IκBα fusion exhibits phosphorylation defects and resistance to TNFα mediated degradation similar to the ones observed for endogenous monoubiquitylated IκBα. The N-terminal attachment of a single ubiquitin moiety on the IκBα fusion results in a deficient binding to the IKKβ kinase and recruitment of the SCF ligase component βTrCP, promoting a negative impact on the NF-κB activity. Altogether, our results suggest the existence of a reservoir of monoubiquitylated IκBα resistant to TNFα-induced proteolysis, which is able to interact and repress DNA binding and NF-κB transcriptional activity. Such pool of IκBα may play an important role in the control of basal and signal-mediated NF-κB activity.


Introduction
The nuclear factor kB (NF-kB) is a family of transcription factors that regulate the expression of various genes involved in inflammatory, anti-apoptotic and immune responses [1] [2]. The NF-kB pathway can be activated by many different extra cellular signals that induce multiple post-translational modifications such as phosphorylation, ubiquitylation and SUMOylation, acting at various levels of the signaling cascade [3][4][5]. As many other stimuli, the pro-inflammatory cytokine TNFa (tumor necrosis factor-alpha) ends with the activation of the IKK (IkBa Kinase) complex, composed by IKKa, IKKb and IKKc/NEMO [6] [7]. IKK phosphorylates the alpha inhibitor of NF-kB, IkBa, on the serines 32 and 36 and targets it for ubiquitylation at the main ubiquitylation sites, lysine 21 and 22 by a SCF (Skp, Cullin, F-box) ubiquitin ligase complex containing the beta-transducin repeatcontaining protein bTrCP) [8] [9]. The presence of the DSGXXS motif determines the specific interaction of bTrCP with the phosphorylated Inhibitor of NF-kB alpha (IkBa), which is crucial for its ubiquitylation and posterior proteasome degradation. In contrast, the conjugation with the small ubiquitin-like modifier 1 (SUMO-1) is not dependent on the phosphorylation on the serines 32 and 36 of IkBa and has a positive impact on IkBa stability [10]. Ubiquitylation of IkBa is tightly controlled by the action of unidentified DUBs (de-ubiquitylating enzymes). Released NF-kB is then imported to the nucleus where it activates the transcription of a large number of genes including IkBa and TNF-receptor 2 [11] [2]. Newly synthesized IkBais imported into the nucleus where it ends up with NF-kB mediated transcription by detaching it from DNA promoter sequences and favoring its export to the cytoplasm [12] [13].
In this study, the use of ubiquitin traps (TUBEs for Tandemrepeated Ubiquitin Binding Entities) [14] allowed us to identify monoubiquitylated IkBa from rat organs, as well as from different cell lines. Using in vitro and ex vivo approaches we aimed to understand the impact that a single ubiquitin moiety can have on the properties and inhibitory capacity of IkBaThe evidence presented here suggests the existence of a pool of monoubiquitylated IkBa resistant to degradation whose function might play an important role in the control of basal and signal-induced NF-kB activity.

Presence of monoubiquitylated IkBa in organs and cell lines
The recently developed ubiquitin-traps (TUBEs) that specifically capture ubiquitin and ubiquitylated proteins [14] were adapted to extract ubiquitylated proteins from rat organs. As reported, TUBEs capture preferentially polyubiquitin proteins, however monoubiquitylated proteins can also be captured when abundantly expressed [14]. Monoubiquitylated IkBa can be easily detected by Western blot in a mix of total ubiquitylated proteins purified by TUBEs from liver, heart, brain, muscle, lung and kidney rat (Figure 1), suggesting a function for this form of IkBa in normal tissues. Monoubiquitylated IkBa can also be captured using a similar procedure with multiple cell lines such as HEK293 (Figure 2A), Jurkat ( Figure 2B) and HeLa (data not shown). The identification of the monoubiquitylated IkBa was confirmed using several protocols including immunoprecipitations with a specific anti-ubiquitin antibody of the TUBE-captured material. Under these conditions endogenous and exogenous monoubiquitylated IkBa can be detected using anti-IkBa, anti HA or anti SV5 antibodies, respectively ( Figure 2C). Furthermore, monoubiquitylated IkBa can be also detected in cells co-transfected with plasmids encoding histidinylated versions of ubiquitin with or without vectors expressing IkBa WT to purify exogenous and endogenous ubiquitylated IkBa respectively ( Figure 2D). Mono-ubiquitylated IkBa can also be easily reproduced in vitro using an ubiquitin mutant (Ub KO) where all reactive lysine residues have been changed to arginine ( Figure 2E). However, monoubiquitylated IkBa cannot be immunoprecipitated with monoclonal or polyclonal IkBa antibodies, alone or combined in a TUBEs-IkBa immunoprecipitation procedure ( Figure S1 and S2). Under these conditions monoubiquitylated IkBa is detected in the unbound fraction. Thus, the monoubiquitylated form of IkBa, found in organs and cell lines, shows a poor accessibility to IkBa immunoprecipitation but can be detected using denaturing gels followed by Western blot analysis.

Monoubiquitylated IkBa is not sensitive to the TNFamediated degradation
To evaluate the susceptibility of the monoubiquitylated form of IkBa to be degraded by TNFa a TUBE-capture assay was performed in HEK293 cells treated or not with proteasome inhibitor MG132 ( Figure 3). As expected, most IkBa is degraded after 20 minutes of TNFa-stimulation as it can be seen in the input (IN). This proteolytical process is blocked in the presence of MG132 where IkBa is accumulated as hyperphosphorylated form. The analysis of the TUBE-captured material shows that monoubiquitylated IkBa remain very stable after 20 or 60 minutes of TNFa stimulation even in the presence of proteasome inhibitor ( Figure 3A). Interestingly, the capacity of the TUBE-hHR23 to capture monoubiquitylated and polyubiquitylated forms of IkBa is not compromised when the proteasome activity is inhibited. These results suggest that the monoubiquitylated form of IkBa is not destabilized by the induction with TNFa but it is slightly accumulated after treatment with MG132 ( Figure 3A). To evaluate the role of the serines 32/36 phosphorylation on the accumulation of monoubiquitylated form of IkBa, a mutant S32/ 36A was transfected into HEK293 cells. In the absence of TNFa stimulation and MG132, modified forms of IkBa were captured Figure 1. Extraction of monoubiquitylated IkBa from rat organs. Extraction of total ubiquitylated proteins from liver, brain, heart, muscle, lung and kidney [14]. TUBEs (T) or GST control (C) bound proteins were analyzed by Western blot with the anti-ubiquitin and anti-IkBa antibodies. Membranes were stained with Ponceau, used as charge control, (1) TUBEs; 2) GST). Input (IN), Flow through (FT). doi:10.1371/journal.pone.0025397.g001 using His6-Ubiquitin, His6-SUMO-1 or His6-SUMO-2 and nickel beads chromatography. Our results confirm that the monoubiquitylation of IkBa is not dependent of the phosphorylation of serines 32 and 36 ( Figure 3B). MonoSUMOylation with SUMO-2 and SUMO-1 (only visible on long exposures, data not shown) are also independent of this signaling pathway. In contrast, high molecular weight forms can not be seen on the S32/36A IkBa mutant after TNFa stimulation in a situation where polyubiquitylated IkBa WT is well accumulated ( Figure 3B).

Extended half-life of monoubiquitylated IkBa
The analysis of a subpopulation of IkBa molecules and in particular its impact on NF-kB activity is difficult to achieve if mixed populations of IkBa molecules are present. There is no available method of purification able to isolate unmodified or ubiquitylated IkBa molecules with homogeneous characteristics. For this reason, to further understand the role of monoubiquitylated form of IkBa, an ubiquitin-IkBa fusion protein was generated ( Figure 4A). This approach has been largely used as it can be judged in the literature especially to study the role of ubiquitin and ubiquitin-like proteins in the regulation of protein localization and function [15]. The ubiquitin-IkBa fusion has been optimized to resist to the action of DUBs by introducing, at the Cterminal of ubiquitin, a double alanine (AA) instead of the double glycine (GG). To avoid additional attachment of moieties at the Nterminus of IkBa, lysine 21 and 22 were mutated to alanine (KK to AA). Attachment of ubiquitin at a single N-terminus lysine acceptor of IkBa provide similar stability effects [16,17].
Ubiquitin-IkBa fusion protein shows similar sub-cellular distribution than IkBa WT ( Figure S3). When expressed in HEK293 and HeLa cells, ubiquitin-IkBa fusion protein showed an extended half-life compared to IkBa WT ( Figure 4B and data not shown). The effect of a single ubiquitin moiety on IkBa stability is also reflected after signal-mediated stimulation, as this ubiquitin-IkBa fusion shows resistance to TNFa induced degradation ( Figure 4C). A kinetic of degradation was performed to confirm that the observed resistance was not due to a delay in TNFa-induced IkBa degradation ( Figure 4D). Proteolytical defects are not due to the Furthermore, these effects appear to be specific of ubiquitin, as fusions containing other molecules from the ubiquitin family do not provide the same results (data not shown). Thus, from these results we conclude that the attachment of a single ubiquitin moiety extends the half-life of the ubiquitin-IkBa fusion and perfectly reproduce the stability after TNFa-stimulation observed with the endogenous monoubiquitylated form of IkBa.

Phosphorylation defects of monoubiquitylated IkBa
In order to understand the molecular origin of ubiquitin-IkBa fusion stability, its capacity to be phosphorylated after TNFa stimulation was investigated. We could observe a reproducible reduction of ubiquitin-IkBa fusion phosphorylation when compared to IkBa WT ( Figure 5A). This is mainly due to the incapacity of ubiquitin-IkBa fusion protein to efficiently bind IKKb compared to IkBa WT in TNFa-stimulated HEK293 cells ( Figure 5B). Experimental data demonstrate that the exogenously expressed ubiquitin-IkBa fusion also fails to efficiently interact with bTrCP compared to IkBa WT under the same experimental conditions. Altogether our results clearly indicate that ubiquitin-IkBa fusion but not IkBa WT shows defects in the interaction with critical molecules of the signaling pathway including IKKb and bTrCP, thus explaining at least in part, its resistance to proteolysis ( Figure 5B).

Monoubiquitylated IkBa negatively affects NF-kB activity
To investigate the effect of ubiquitin-IkBa fusion on the NF-kB activity, first we explored its capacity to bind NF-kB and to inhibit NF-kB/DNA binding. Interaction with the NF-kB subunit p65 was tested in HEK293 cells expressing or not exogenous p65 and ubiquitin-IkBa or IkBa WT as indicated in Figure 6A. Our results clearly indicate that p65 co-immunoprecipitates equally well with both ubiquitin-IkBa and IkBa WT. To explore the capacity of ubiquitin-IkBa fusion to inhibit NF-kB/DNA binding, electrophoretic mobility shift assay (EMSA) were performed using increasing Figure 6. Ubiquitin-IkBa fusion negatively affects TNF-induced NF-kB activity. A) Ubiquitin-IkBa fusion protein has the same capacity as unmodified IkBa to bind to NF-kB. HEK293 cells were transfected with the indicated plasmids. Lysates were submitted to anti-HA immunoprecipitation and Western blotted with the indicated antibodies B) Ubiquitin-IkBa fusion protein inhibits NF-kB/DNA binding as well as unmodified IkBa. Different amount of recombinant IkBa fusions proteins (1: 0,05 ml, 2: 0,1 ml and 3: 0,5 ml) and p65 were incubated with a radioactive labeled NF-kB probe for EMSA studies [12]. Comp.: competition with a 100-fold excess of the same unlabeled oligonucleotide added to the binding assay before the 32 P-labeled probe. The graph corresponds to the mean of three independent experiments. C) IkBa KO fibroblasts were cotransfected with IkBa WT or ubiquitin-IkBa fusion expressing plasmid and a NF-kB-luciferase reporter (3 EnhConA-Luc). Luciferase activity was measured as previously described [17]. The graph corresponds to the mean of three independent experiments. doi:10.1371/journal.pone.0025397.g006 concentrations of both ubiquitin-IkBa and IkBa WT. As observed in Figure 6B, the capacity of ubiquitin-IkBa fusion to inhibit NF-kB/DNA interaction is similar to the one of IkBa WT. To further explore the effect of ubiquitin-IkBa on the NF-kB activity after TNFa stimulation, luciferase reporter assays were set up. To avoid interferences with endogenous IkBa MEF coming from IkBa KO mice were employed. The inhibitory effect of ubiquitin-IkBa fusion became statistically significant after 6 hours of TNFa stimulation ( Figure 6C). Thus, our results indicate that in the presence of ubiquitin-IkBa fusion, activation of NF-kB is negatively regulated after TNFa stimulation. Altogether, our results suggest the existence of distinct populations of IkBa molecules among which monoubiquitylated IkBa offers resistance to TNFa mediated degradation preserving a dormant pool of NF-kB that is different to the one activated through the activation of this signaling pathway (Figure 7).

Discussion
While polyubiquitylation of proteins has been associated to the regulation of signaling cascades or protein degradation by the Ubiquitin Proteasome System, monoubiquitylation or multiple monoubiquitylation have diverse non-catabolic functions [18]. Major technical problems to separate the ubiquitylated pool of endogenous proteins from unmodified ones justify our limited knowledge of the post-modification events. The broad distribution of monoubiquitylated IkBa in multiple organs and cell lines underlines the in vivo importance of this pool of IkBa. Endogenous monoubiquitylated IkBa is stable after TNFa stimulation and does coexist with polyubiquitylated IkBa under the same conditions. Therefore monoubiquitylated IkBa does not appear to be a precursor of polyubiquitylated forms of this inhibitor molecule, although a dynamic equilibrium between these populations cannot be excluded. Monoubiquitylated IkBa accumulated after proteasome inhibitor and TNFa treatment can be the result of proofreading mechanism acting on polyubiquitylated IkBa. With the help of a DUB-resistant ubiquitin-IkBa fusion, results presented here show that monoubiquitylated IkBa has an impact on basal and TNFa-induced NF-kB transcription. It remains to be investigated the nature of the stimuli (if any) able to drive an efficient proteolysis of monoubiquitylated IkBa. In a similar way SUMO-1 was reported to regulate IkBa stability and NF-kB transcription [10]. However it is unclear if both IkBa pools cooperate with each other to regulate basal and/or signal mediated turnover. Several lines of evidence suggest that monoubiquitylated IkBa might adopt a structured/protected conformation. The first evidence is the difficulty to pull down endogenous monoubiquitylated IkBa with various monoclonal and polyclonal antibodies. Second, the ubiquitin-IkBa fusion is not efficiently recognized by the IKKb subunit resulting in a limited phosphorylation and binding to bTrCP. Finally, the impact of the fused ubiquitin on the IkBa resistance to TNFainduced proteolysis, suggest reduced capacity to interact/get access to the proteasome. Attachment of monoubiquitin onto IkBa perhaps occludes IKK binding sites or creates molecular interference with this kinase. Under these circumstances, bTrCP might have difficulties to polyubiquitylate poorly phosphorylated IkBa. The unstructured extremities of IkBa favor the ubiquitinindependent proteasomal degradation of this molecule [19] justifying the necessity to generate a stable pool of monoubiquitylated IkBa. If there is an ubiquitin-protein ligase different than the SCF-bTrCP complex, it has to be proven. However, one has to keep in mind that E3-independent monoubiquitylation has been reported for proteins containing ubiquitin-binding domains [20]. Recently, monoubiquitylation of Rpn10 subunit of the proteasome has been shown to adopt a closed conformation due to the intramolecular interaction with its ubiquitin interacting motif or UIM [21]. In the case of Rpn10, monoubiquitylation affects presentation of ubiquitylated proteins to the proteasome. However, if this occluding mechanism exists for monoubiquitylated IkBa there is no evidence of an ubiquitin-binding domain present on this inhibitor. Nonetheless, the active molecular dynamics reported for the NF-kB system even under basal conditions [22] [23] [24] justifies the existence of monoubiquitylated IkBa as a cellular reservoir to regulate basal as well as signal-mediated NF-kB activity. Knowing the resistance to proteolysis observed for monoubiquitylated IkBa, one can speculate that an artificial increase of this pool could let to a better control of immune and/or pro-inflammatory responses found in organisms that have been exposed to multiple and/or sequential stimuli activating NF-kB. Future work will elucidate the role of the different populations of IkBa in the optimal control of this critical transcription factor.

Animals
Ethics Statement. Experiments were approved by the respective institutional committees for animal care and handling.
Adult male Sprague-Dawley rats were deeply anesthetized with chloral hydrate and some tissues and organs were extracted. These samples were washed with cold PBS, immediately frozen in liquid nitrogen and stored at 280uC.

Cell cultures
HEK293 and HeLa (ATCC) were grown in DMEM (Gibco); Jurkat cells (ATCC) in RPMI (Gibco), all supplemented with 10% FBS and antibiotics. HEK293 and HeLa were transfected using lipofectamine (Invitrogen). For measurement of transcriptional activity, MEF null IkBa (kindly given by David Baltimore) were co-transfected with a NF-kB-luciferase reporter plasmid (3-EnhConA) and plasmids expressing IkBa WT or the ubiquitin-IkBa fusion. Luciferase activity was measured as previously described [17]. To analyze the half-life of the different proteins, cells were treated with 50 mg/ml of cycloheximide (Sigma) during the indicated times. For stability experiments, cells were treated for 1 hour with 20 mM of MG132 (Calbiochem), stimulated for 30 minutes or the indicated times with 10 ng/ml of TNFa(R&D Systems). P65, IKKb and bTrCP co-immunoprecipitation experiments were performed using Protein-G cross-linked with the HA antibody to immunoprecipitate exogenous IkBa WT or ubiquitin-IkBa fusion protein. In all cases, cells were lysed for 15 minutes on ice in 50 mM sodium fluoride, 5 mM tetra-sodium pyrophosphate, 10 mM beta-glyceropyrophosphate, 1% Igepal CA-630, 2 mM EDTA, 20 mM Na 2 HPO 4 , 20 mM NaH 2 PO 4 , 1 mM Pefablock, 1.2 mg/ml Complete protease inhibitor cocktail (Roche).

PCR and cloning
Ubiquitin gene (accession numbers CAA44911) was used to generate IkBa fusion and cloned into BamHI/Not1 restriction sites of pCDNA3. The C-terminal glycine residues (GG) of ubiquitin were changed to alanine (AA) and lysine 21 and 22 of IkBa were mutated to alanine to avoid respectively the action of DUBs and additional attachment of moieties at the N-terminus of IkBa, using the following oligonucleotides: 59-ctc cgt ctt aga gct gcg gag cgg cta ctg gac gac-39 and 59-gtc gtc cag tag ccg ctc cgc agc tct aag acg gag-39. His6-Ubiquitin construct has been previously reported [13].

Purification of ubiquitylated proteins
Frozen tissues were triturated in liquid nitrogen and recovered in the previously reported lysis buffers [14], containing 200 mg of TUBEs-HR23A (T) (Life Sensors) or Glutathione S-transferase (GST) (C). Lysates were clarified by cold centrifugation, and added to glutathione agarose beads (Sigma). Glutathione beads were eluted and bound material was submitted to Western-blot analysis or to IkBa (Cell Signaling) and ubiquitin (FK2, ENZO) immunoprecipitations. His6-ubiquitylated proteins were purified using denaturing conditions and Ni 2+ chromatography as previously described [13].

In vitro ubiquitylation assays
In vitro transcribed/translated IkBa ( 35 S-Met-labelled or not when indicated) were incubated in a 15 ml reaction including an ATP regenerating system [25 mM Tris pH 7.5, 5 mM MgCl2, 2 mM ATP, 10 mM creatine phosphate (Sigma), 5 mM NaCl 2 , 3.5 U/ml of creatine kinase (Sigma) and 0.6 U/ml of inorganic pyrophosphatase (Sigma)], 10 mg of ubiquitin mutant (Ub KO) where all reactive lysine residues have been changed to arginine ubiquitin, 10 ng human E1 (Biomol), 500 ng UbcH5b (Biomol). After incubation at 30uC for 120 min the reaction was stopped with SDS Laemmli buffer containing b-mercaptoethanol, samples were fractionated by SDS-PAGE and the dried gels analysed by phosphorimaging.

Electrophoretic Mobility Shift Assays (EMSA)
Reactions were prepared in binding buffer containing 25 mM HEPES, 1 mM EDTA, 3.5 mM spermidine, 6 mM MgCl2, 100 mM NaCl, 0.15% Nonidet P-40, 10% glycerol, 10 mM Dithiothreitol, 1 mg/ml bovine serum albumin and 0.05 mg Poly dAT/dGC, different amount of IkBa fusion proteins (1: 0,05 ml, 2: 0,1 ml and 3: 0,5 ml) and recombinant protein p65 and incubated at room temperature for 20 minutes. Finally, 10000 cpm of 32 Pradiolabelled (polynucleotide kinase, Biolabs) double strand oligonucleotide probe containing the NF-kB binding site motif from the HIV type 1 enhancer (59-CTA GAC GGG GAT TTC CGA GAG GT-39) was added and the mixture was incubated at room temperature for 20 minutes. After electrophoresis, gels were dried and exposed to Amersham Hyperfilm MP at 270?C. Specific binding was checked by competition with a 100-fold excess of the same unlabeled oligonucleotide added to the binding assay before the 32 P-labeled probe. Figure S1 Immunoprecipitation using IkBa antibodies fail to pull down monoubiquitylated IkBa. HEK293 cells were treated or not for 1 hour with 20 mM of MG-132, lysed in the properly lysis buffer for 20 minutes, centrifuged and the supernatant was incubated with cross-linked anti-IkBa (10B) antibody for 2 hours. After incubation the samples were centrifuged, washed and prepared for Western blot analysis using IkBa antibody (Cell Signaling). (TIF) Figure S2 TUBE-captured monoubiquitylated IkBa fails to be immunoprecipitated using specific IkBa antibodies. HEK293 cells were treated or not, 1 hour with 20 mM of MG-132 and lysed in a buffer containing 100 mg of TUBE-HR23A or GST proteins. After lysis, samples were centrifuged and clarified supernatant incubated for 2 hours in the presence of glutathione agarose beads. Eluted samples were incubated for 2 hours with protein A cross-linked antibody anti-IkBa 10B or anti-IkBa C21 antibody (not shown). After incubation, samples were washed and prepared for Western blot analysis using IkBa antibody (Cell Signaling). (TIF) Figure S3 IkBaWT and ubiquitin-IkBa fusion were expressed in HEK293 cells, and processed for immunostaining with anti-SV5 or anti-HA antibodies. (TIF)