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Nuclear Accumulation of cRel following C-Terminal phosphorylation by TBK1/IKK¹

Terry Fox Molecular Oncology Group, Lady Davis Institute for Medical Research, and Department of Microbiology & Immunology, Department of Medicine and Department of Oncology, McGill University, Montréal, Québec, Canada; and Department of Biochemistry, Centre de Recherche du CHUM, Faculty of Medicine, University of Montréal, Montréal H2X 1P1, Québec, Canada

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The NF-B transcription factors are key regulators of immunomodulatory, cell cycle, and developmental gene regulation. NF-B activity is mainly regulated through the phosphorylation of IB by the IB kinase (IKK) complex IKK, leading to proteasome-mediated degradation of IB, nuclear translocation of NF-B dimers, DNA binding, and gene induction. Additionally, direct posttranslational modifications of NF-B p65 and cRel subunits involving C-terminal phosphorylation has been demonstrated. The noncanonical IKK-related homologs, TNFR-associated factor family member-associated NF-B activator (TANK)-binding kinase (TBK)1 and IKK, are also thought to play a role in NF-B regulation, but their functions remain unclear. TBK1 and IKK were recently described as essential regulators of IFN gene activation through direct phosphorylation of the IFN regulatory factor-3 and -7 transcription factors. In the present study, we sought to determine whether IKK and TBK1 could modulate cRel activity via phosphorylation. TBK1 and IKK directly phosphorylate the C-terminal domain of cRel in vitro and in vivo and regulate nuclear accumulation of cRel, independently of the classical IB/IKK pathway. IB degradation is not affected, but rather IKK-mediated phosphorylation of cRel leads to dissociation of the IB-cRel complex. These results illustrate a previously unrecognized aspect of cRel regulation, controlled by direct IKK/TBK1 phosphorylation.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The NF-B transcription factors are key regulators of genes involved in inflammatory and immunomodulatory responses, cell proliferation, and cell survival (reviewed in Refs. 1 and 2). Five mammalian members have been identified: p50 (NF-B1), p52 (NF-B2), p65 (RelA), cRel, and RelB. All members share similar structural elements including a Rel homology domain, which is composed of a dimerization domain, a nuclear localization signal, a DNA-binding domain, in the N-terminal extremity, as well as a serine-rich transactivation domain (TD)⁴ in the C-terminal end. Although p50, p52, and p65 are expressed in virtually all cell types, RelB is restricted to lymphoid cells and cRel expression is limited to the hemopoietic lineage (3, 4). Studies in cRel^–/– cells revealed that cRel plays an essential role in the induction of a proper lymphocyte response to mitogen stimulation and in the production of cytokines such as IL-2, IL-3, and GM-CSF (5, 6, 7).

In unstimulated cells, cytoplasmic NF-B subunits interact with the inhibitory IB molecules. The IB-NF-B complexes shuttle between the nucleus and the cytoplasm with an equilibrium in favor of the cytoplasm (1). Following stimulation by proinflammatory cytokines IL-1 or TNF-, phosphorylation of the IB inhibitor by the multimeric IB kinase (IKK) complex leads to IB polyubiquitination and subsequent degradation by a proteasome-dependent pathway, liberation of NF-B dimers, nuclear translocation, and transcription of NF-B target genes. Thus, the IKK complex, composed of two catalytic subunits IKK and IKK and of a regulatory subunit IKK/NEMO (8, 9, 10), is a central component of the classical NF-B activation cascade.

Recently, new members of the IKK-related kinase family, TNFR-associated factor (TRAF) family member-associated NF-B activator (TANK)-binding kinase (TBK)1 (also known as T2K or NAK) and IKK (initially isolated as IKKi), were identified (11, 12, 13, 14). IKK and TBK1 are structurally similar to the classical IKK kinases, with an N-terminal kinase domain and C-terminal leucine zipper-like and helix-loop-helix domains but share only 27% primary sequence identity with IKK, whereas IKK and TBK1 possess 61% sequence identity to each other and are enzymatically similar (15). In contrast to the role of the IKK complex in the phosphorylation of IB at Ser³² and Ser³⁶, TBK1 and IKK phosphorylate IB solely on Ser³⁶ (11) and the physiological significance of this phosphorylation in NF-B activation remains unclear. However, a distinct role for IKK and TBK1 in NF-B activation was suggested by the observation that a kinase inactive, dominant negative form of IKK, IKK(K38A), blocked NF-B induction in response to PMA or TCR stimulation, but not in response to TNF- or IL-1 cytokines (11). Similarly, dominant negative TBK1 inhibited the effect of TANK/I-TRAF on NF-B activation (13). Furthermore, IKK was reported to activate NF-B through direct phosphorylation of TANK/I-TRAF and subsequent liberation of TRAF2 (16). In tbk1^–/– cells, IB degradation and NF-B binding was intact in response to IL-1 and TNF- stimulation but NF-B transcriptional activity was defective (14).

In addition to their partially defined roles in NF-B activation, TBK1 and IKK are key components in the development of the IFN-mediated antiviral response (17, 18). Following virus infection or TLR3 or TLR4 engagement, TBK1 and IKK phosphorylate distinct serine residues in the C-terminal regulatory domains of transcription factors IFN regulatory factor (IRF)-3 and IRF-7, leading to dimerization, nuclear translocation, DNA binding, and IFN gene activation (17, 19, 20), thereby permitting the establishment of an antiviral state.

Direct modulatory phosphorylation and acetylation of NF-B subunits are important additional steps in the NF-B activation cascade (reviewed in Ref. 21). Thus far, inducible phosphorylation of RelA/p65 at serine residues 276 or 311 in the Rel homology domain or serine residues 468, 529, and 536 in the TD have been observed following exposure of cells to various NF-B-inducing agents, culminating in the modulation of nuclear localization, DNA-binding affinity, coactivator/corepressor association, and transactivation capacity (14, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33). Inducible phosphorylation of cRel was also detected in response to PMA, CD28, or TNF- treatment of T cells (34, 35, 36); phosphorylation of Ser⁴⁷¹ in the TD has been reported to be involved in TNF--induced activation of cRel (34, 35), whereas Ser⁴⁵¹ in the TD of murine cRel serves as a phospho-acceptor site, although the physiological function of this event remains to be elucidated (37).

Given the important function of IKK and TBK1 in the direct phosphorylation of IRF-3 and IRF-7, we examined the possibility that IKK and TBK1 may modulate cRel activity through direct phosphorylation. We now demonstrate that IKK phosphorylates cRel in vitro and in vivo within two regions of the TD and induces cRel nuclear accumulation in a manner that is independent of the classical IKK-IB pathway. IB degradation is not affected, but rather IKK-mediated phosphorylation of cRel leads to dissociation of the IB-cRel complex. These results illustrate that cRel function is regulated by direct IKK- and TBK1-mediated phosphorylation and reveal a previously unrecognized aspect of NF-B regulation by IKK and TBK1.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Plasmids

The pcDNA3.1zeo-flag-IKKwt, pcDNA3.1zeo-flag-IKKK38A, pcDNA3.1zeo-flag-TBK1wt, pcDNA3.1zeo-flag-TBK1K38A, SVK3-IBwt, and SVK3-IB2N constructs were previously described (17, 38). The pcDNA3.1zeo-flag-IB2N plasmid was generated by subcloning the IB2N cDNA from the SVK3-IB2N constructs. Plasmids encoding Myc-IKK, Flag-IKKwt, and Flag-IKKDN were previously described (39). The pcDNA3.1zeo-myc-cRel plasmid was generated by cloning the PCR-amplified forward primer 5'-ATA TAA GCT TAG CGG AGC CAT GGC CTC CGG TGC GTA TAA-3', reverse primer 5'-ATC GGA ATT CTA CAA AAT GCT GCA TCT ATA T-3' cRel sequence into the EcoRI/HindIII sites of the pcDNA3.1zeo-Myc vector. The cRelTD-pcDNA3.1zeo-Myc vector construct was generated by PCR amplification of the cRelTD cDNA forward primer 5'-ATA TAA GCT TAG CGG AGC CAT GGC CTC CGG TGC GTA TAA-3', reverse primer 5'-GAA TTC TCA ATT CCC AAC AGG TAT TCT-3', followed by subcloning into the EcoRI/HindIII sites of the Myc-pcDNA3.1zeo vector. The GST-cRelTD (aa 422–587), GST-cRelA (aa 422–472), GST-cRelB (aa 473–531), and GST-cRelC (aa 532–587) encoding constructs were generated by PCR amplification, using the pCMVBL-cRel plasmid as a template (40) and subcloning into the pGEX4T2 vector using the BamHI/EcoRI restriction sites. The GST-IB (aa 1–55) encoding construct was previously described (17). The IRF-4prom-Luc construct containing the CD28RE region (nt –367 to +1) of the IRF4 promoter was previously described in (41).

Cell culture and transfection

The HEK 293T cells (American Type Culture Collection (ATCC)) were cultured in DMEM (Wisent) containing 10% heat-inactivated FCS, and antibiotics. Subconfluent 293T cells were transfected using the calcium phosphate precipitation method. HeLa cells (ATCC) were grown in DMEM containing 10% FCS and antibiotics and transfected with Fugene reagent according to the manufacturer’s instructions (Roche).

Reporter gene assays

HEK 293T cells were transfected in 24-well plates with a constant amount of IRF-4prom-Luc construct (200 ng) together with 20 ng of pRLTK plasmid as internal reference, 2 µg of pcDNA3.1zeo-myc-cRel plasmid and an increasing amount (0–500 ng) of pcDNA3.1zeo-Flag-IKKwt plasmid. At 24 h posttransfection, the luciferase activity was measured using the dual luciferase kit following the manufacturer’s instructions (Promega). Relative luciferase activities were calculated as luciferase to Renilla ratio. Results were then expressed as fold compared with the empty vector.

Immunoblot analysis

Whole cell extracts prepared as previously described (42) were resolved by SDS-PAGE and transferred onto nitrocellulose membrane (Bio-Rad). The membrane was blocked in PBS containing 0.05% Tween 20 and 5% nonfat dry milk, or 5% BSA for phospho-specific Abs, for 1 h and incubated with primary Ab, anti-cRel (1/1000) (43) or anti-cRel sc-70 from Santa Cruz Biotechnology (1 µg/ml), anti-IB MAD3-10B (1/1000), anti-IB phospho-Ser³² (1/1000; Cell Signaling Technology), anti--actin (Chemicon International), anti-Flag M2 (1 µg/ml; Sigma-Aldrich), anti-Myc 9E10 (1 µg/ml; Sigma-Aldrich), and anti-IKK (2 µg/ml; Imgenex), in blocking solution. After five 5-min washes in PBS containing 0.05% Tween 20, the membranes were incubated for 1 h with HRP-conjugated goat anti-rabbit or goat anti-mouse IgG (1/2000 to 1/10000; Kirkegaard & Perry Laboratories) in blocking solution. Immunoreactive proteins were visualized by ECL (PerkinElmer).

Metabolic labeling

At 21 h posttransfection, 293T cells were washed twice in phosphate-free DMEM (MP Biomedicals) and further cultured in phosphate-free DMEM supplemented with 1 mM glutamine, 10% dialyzed FBS, and 0.2 mCi/ml [³²P]orthophosphoric acid. After 3 h, cells were washed in cold PBS, lysed in lysis buffer, and subjected to immunoprecipitation using protein G-Sepharose beads precoupled with anti-Myc Abs (1 µg). Immunocomplexes were resolved by SDS-PAGE and transferred on nitrocellulose membrane. Incorporation of ³²P was revealed by autoradiography. Immunoprecipitation of Myc-cRel was revealed by immunoblot analysis using anti-cRel Abs as described.

In vitro kinase assay

Recombinant GST-fusion proteins used as substrates were produced in Escherichia coli BL21 bacteria. The production of the recombinant protein was induced with 1 mM IPTG (isopropyl--D-thiogalactopyranoside) for 3 h at 37°C. After induction, bacterial pellet was lysed in PBS with 1% Triton X-100 and sonicated for 6 min by pulses of 10 s on, 10 s off at 30% efficiency. Bacterial lysate was cleared by centrifugation for 15 min at 10,000 rpm at 4°C. GST-peptides were then purified from the cleared bacterial lysate by pulldown with glutathione Sepharose beads. Recombinant IKK, IKK, and IKK kinases were produced using an in vitro rabbit reticulocytes lysate transcription/translation system using ³⁵S-labeled methionine according to the manufacturer’s protocol (TNT T7-coupled rabbit reticulocytes system; Promega). Recombinant TBK1 was produced in the baculovirus/insect cell system and purified as previously described (19). Recombinant kinases were immunoprecipitated with protein G-Sepharose beads precoupled to anti-Myc or anti-Flag Abs. Immunocomplexes were washed twice in lysis buffer and twice in IKK buffer (20 mM HEPES, 150 mM NaCl, 20 mM MgCl₂, 1 mM DTT, 0.1 mM sodium orthovanadate, 20 mM glycerophosphate, and 10 mM p-nitrophenylphosphate) and used in the kinase reaction. The kinase reaction was performed by incubation of the immunocomplexes with 10 µCi [-³²P]ATP, 20 µM ATP, 1.0–3.0 µg of GST substrate at 30°C for 30 min in kinase buffer. Proteins were resolved by 10% acrylamide SDS-PAGE. The gel was stained with Coomassie blue for 15 min, destained in 10% ethanol-10% acetic acid, dried and revealed by exposition to Biomax XR film (Kodak).

Immunofluorescence

For immunofluorescence analyses, HEK 293T or HeLa cells were grown on coverslips and transfected with the indicated expression plasmids as described. At 24 h posttransfection, cells were fixed in a mixture of methanol to acetone (1:1) for 1 min at room temperature, washed in PBS, and incubated with anti-Flag M2-FITC (20 µg/ml; Sigma-Aldrich) and/or anti-Myc Cy3 conjugate (1/1000; Sigma-Aldrich) in PBS at room temperature in the dark for 1 h. After, two washes in PBS, the cells were incubated in 1 µg/ml Hoescht 33342 in PBS for 2 min. The cells were then subjected to three washes in PBS and a fourth wash in water and mounted on slides with ProLong anti-fade mounting media (Molecular Probes). Fluorescence was visualized using an epifluorescence Olympus microscope and pictures analyzed with the ImagePro (Media Cybernetics) software.

Coimmunoprecipitation of the cRel-IB complex

For immunoprecipitation experiments, HEK 293T cells were transfected with a 1:1 ratio of pcDNA3.1zeo-Myc-cRel to pcDNA3.1zeo-flag-IB2N plasmids in the absence or presence of pcDNA3.1zeo-Flag-IKKwt as indicated. Protein G-Sepharose beads (Sigma-Aldrich) were incubated with 1 µg of anti-Myc Abs for 1 h in TNET buffer (50 mM Tris-HCl (pH 7.4), 100 mM NaCl, 2 mM EDTA, 0.1% Triton X-100) containing 1% BSA and washed two times in lysis buffer. Then, whole cell extract (250 µg) prepared as described was incubated with the beads for 3 h at 4°C and immunocomplexes were eluted in Laemmli loading buffer, heated at 100°C, resolved by SDS-PAGE and transferred on nitrocellulose membrane. Immunodetection was performed with anti-Myc and anti-Flag Abs as described.

Preparation of nuclear and cytoplasmic extracts

HEK 293T cells were harvested and washed two times in cold PBS. The cell pellets were resuspended in TKM buffer (1 mM Tris (pH 7.6), 10 mM KCl, 5 mM MgCl₂, 0.2% Nonidet P-40) supplemented with 5 µg/ml leupeptin and 5 µg/ml aprotinin and kept on ice for 20 min and vortexed briefly. Nuclei were pelleted at 3000 rpm for 5 min in a microcentrifuge. The supernatant was further centrifuged at 13,000 rpm for 25 min and the supernatant was considered as the cytoplasmic fraction. The nuclei were washed two times with a 10x volume of TKM buffer to eliminate cytoplasmic contamination, resuspended in lysis buffer containing 5 µg/ml leupeptin and 5 µg/ml aprotinin, and submitted to three freeze/thaw cycles. Subcellular extract purity was verified by immunoblot, using anti-poly(ADP-ribose) polymerase (1/1000; BD Biosciences) and anti-tubulin (1 µg/ml; Santa Cruz Biotechnology) Abs as described.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
IKK induces cRel phosphorylation in vivo

First, to determine whether cRel was phosphorylated in response to IKK expression, HEK 293T cells were cotransfected with Myc-tagged cRel, Flag-tagged wtIKK, or IKK(K38A) encoding plasmids and subjected to ³²P-metabolic labeling. IKK expression (Fig. 1A, lane 2), but not expression of the inactive kinase IKK(K38A) (Fig 1A, lane 4), resulted in a significant increase in ³²P incorporation into cRel, compared with the empty vector control (Fig. 1A, lanes 1 and 3), thus demonstrating an IKK-inducible phosphorylation of cRel. By immunoblot analysis, a slower migrating form of cRel was detected in extracts containing increasing amounts of IKK (Fig. 1B, lanes 4 and 5), but not in extracts from cells expressing IKK(K38A) mutant (Fig. 1B, lanes 7–10). Similarly in HeLa cells, IKK expression correlated with the detection of a slower migrating form of cRel (Fig. 1B, lane 12), compared with either control cells (Fig. 1B, lane 11) or IKK(K38A) expressing cells (Fig. 1B, lane 13). These results indicate that cRel is phosphorylated in vivo in response to IKK expression.

View larger version (32K): [in this window] [in a new window]   FIGURE 1. IKK induces cRel phosphorylation in vivo. A, HEK 293T cells were cotransfected with Myc-tagged cRel encoding constructs together with the control vector (lane 1 and lane 3), Flag-tagged IKKwt (lane 2), or Flag-tagged IKK(K38A) (lane 4) encoding plasmids. Transfected cells were subjected to 32P metabolic labeling and cRel was immunoprecipitated from whole cell extract using anti-Myc Abs. Immunocomplexes were resolved by SDS-PAGE and radioactivity incorporation was measured by autoradiography. B, HEK 293T cells or HeLa cells were transfected with a plasmid encoding Myc-tagged cRel together with various amounts of Flag-tagged IKKwt (lanes 2–5 and lane 12) or Flag-tagged IKK(K38A) (lanes 7–10 and lane 13) encoding plasmid. At 24 h posttransfection, Whole cell extracts were prepared and resolved by SDS-PAGE and proteins were revealed by immunoblot using anti-cRel, anti-Flag, or anti-actin Abs.

IKK directly phosphorylates the cRel TD in vitro

Next, to determine whether cRel phosphorylation was the direct result of IKK activity, in vitro kinase assays were performed using in vitro transcribed and translated Flag-tagged wtIKK (Fig. 2, lanes 4–6), Flag-tagged IKKwt (Fig. 2, lanes 7–9), Myc-tagged IKKwt (Fig. 2, lanes 10–12) and purified GST-cRel TD fusion protein, containing the cRel TD (aa 422–587) fused to GST as a substrate. wtIKK (Fig. 2, lane 5) and wtIKK (Fig. 2, lane 8), but not wtIKK (Fig. 2, lane 11), directly phosphorylated GST-cRel TD; all kinase activities were also able to phosphorylate the GST-IB fusion protein (Fig. 2, lanes 4, 7, and 10), but not the GST protein alone (Fig. 2, lanes 6, 9, and 12) Moreover, the IKK(K38A) dominant negative kinase inactive form was unable to phosphorylate GST-cRel TD (data not shown) indicating that IKK, as well as IKK, but not IKK, target the TD of cRel for direct phosphorylation.

View larger version (50K): [in this window] [in a new window]   FIGURE 2. IKK and IKK directly phosphorylate the transactivation domain of cRel in vitro. Flag-IKK (lanes 4–6), Flag-IKK (lanes 7–9) and Myc-IKK (lanes 10–12) were in vitro transcribed or translated in rabbit reticulocyte lysates and immunoprecipitated (IP) with anti-Flag or anti-Myc Abs. In vitro kinase assays were performed with the immunoprecipitated kinases and purified GST-IB (lanes 1, 4, 7, and 10), GST-cRel TD (aa 422–587) fusion protein (lanes 2, 5, 8, and 11), or GST alone (lanes 3, 6, 9, and 12). Substrates were stained with Coomassie blue (CB) and 32P incorporation (KA) was measured by autoradiography. *, phosphorylated cRel; **, phosphorylated IB.

IKK phosphorylates two different regions of the cRel TD

A previously documented consensus target motif for IKK and TBK1 in IB, IRF-3 and IRF-7 substrates corresponds to a MAPK recognition motif, Ser-X-X-X-Ser, where the last serine residue is the phosphoacceptor site (11, 12, 15, 19, 44). Analysis of the amino acid sequence of the cRel TD revealed the existence of three potential IKK and TBK1 consensus sequences. To determine which of these sites were directly targeted by IKK, three peptides of the cRel TD, aa 422–472 (cRel A), aa 473–531 (cRel B), and aa 532–587 (cRel C), each containing only one consensus site (Fig. 3A), were fused to GST and used in an in vitro kinase assay with recombinant wtIKK (Fig. 3B, lanes 7–12), wtIKK (Fig. 3B, lanes 13–18) and wtIKK (Fig. 3B, lanes 19–24). cRel A (Fig. 3B, lane 20) and cRel C (Fig. 3B, lane 22), but not cRel B (Fig. 3B, lane 21), were phosphorylated by IKK, whereas regions B and C (Fig. 3B, lane 15 and lane 16), but not region A (Fig. 3B, lane 14) were phosphorylated by IKK. As expected from the result, IKK did not phosphorylate any of the peptides (Fig. 3B, lanes 8–10). Thus, IKK targets serine residues within region A and C of the cRel TD domain, whereas IKK targets serines in region B and C.

View larger version (65K): [in this window] [in a new window]   FIGURE 3. IKK phosphorylates cRel in region A and region C in the transactivation domain. A, Schematic representation of cRel amino acid sequence showing smaller peptides cRel A (aa 422–472), cRel B (aa 473–531), and cRel C (aa 532–587), each containing one potential consensus site (underlined sequence) for IKK and TBK1 phosphorylation, designed in the transactivation domain. B, In vitro transcribed or translated Myc-IKK (lanes 7–12), Flag-IKK (lanes 13–18), Flag-IKK (lanes 19–24) were immunoprecipitated (IP) with anti-Flag or anti-Myc Abs. In vitro kinase assays were performed with the immunoprecipitated kinases and purified GST-cRel TD, GST-cRel A, GST-cRel B, GST–cRel C, and GST-IB (aa 1–55) (positive control) fusion proteins or GST alone (negative control). Substrates were stained with Coomassie blue (CB) and 32P incorporation (KA) was measured by autoradiography. Kinases were detected by the incorporation of 35S used in the in vitro translation. Arrows indicate the phosphorylated cRel. *, phosphorylated cRel; **, phosphorylated IB.

IKK expression triggers cRel nuclear translocation

Because cytoplasmic to nuclear translocation is one of the first manifestations of NF-B activation, the effect of IKK expression on cRel subcellular localization was evaluated. Myc-tagged cRel and IB encoding constructs (1:1 ratio), together with Flag-tagged wtIKK or IKK(K38A) encoding plasmids, were expressed in HEK 293T cells and at 24 h posttransfection, indirect immunofluorescence staining of cRel and/or IKK was performed using Cy3-labeled anti-Myc and FITC-labeled anti-Flag Abs, respectively. As shown in Fig. 4A, cRel was detected in the cytoplasm in 91% of control cells (empty vector), whereas in wtIKK-expressing cells, cRel exhibited exclusive nuclear staining in 94% of the cells. Importantly, the localization of cRel remained exclusively cytoplasmic in 88% of IKK(K38A)-expressing cells. The same results were obtained in HeLa cells (Fig. 4B).

View larger version (38K): [in this window] [in a new window]   FIGURE 4. IKK activity induces cRel nuclear accumulation. HEK 293T cells (A) or HeLa cells (B) were cotransfected with plasmids encoding Myc-tagged cRel, IB, and the control, Flag-IKKwt-, or flag-IKKK38A-expressing plasmids. Cells were fixed and stained with Cy3-labeled anti-Myc (red) or FITC-labeled anti-Flag (green), and nuclei were revealed with Hoechst 33342 (blue). Percentage of cells expressing cRel in the nucleus was determined by counting cells expressing Myc-cRel and Flag-IKK (where transfected) and is expressed as the mean of four fields ± SE. Four x40 fields were counted for each condition. Data are representative of at least six independent experiments. C, 293T cells were cotransfected with IRF-4prom-Luc construct and plasmids encoding Myc-tagged cRel () or GFP as control () with increasing amount of Flag-tagged IKKwt encoding plasmid. pRL-thymidine kinase plasmid (Renilla luciferase) was cotransfected and used as an internal control. After 24 h of transfection, the luciferase activity was measured and expressed as fold activation over the transfection of cRel with empty vector after normalization with Renilla luciferase activity. Each value represents the mean ± the SE of triplicate independent samples. The data are representative of three different experiments with similar results.

TBK1 also triggers cRel nuclear translocation

Given the structural and functional similarity between IKK and TBK1, we next sought to determine whether TBK1 could also directly phosphorylate cRel and mediate nuclear accumulation of the protein. Increasing amounts of TBK1 was indeed able to stimulate cRel phosphorylation, based on the slower migration of cRel in SDS-PAGE (Fig. 5A, lane 6). Likewise, baculovirus expressed and purified TBK1 phosphorylated cRel A and cRel C peptides, but not cRel B (Fig. 5B), as shown for IKK. Finally, coexpression of Myc-tagged cRel, together with GFP-tagged TBK1 in HeLa cells, resulted in cRel nuclear translocation in >90% of the transfected cells, whereas cRel remained exclusively cytoplasmic in >85% of GFP-TBK1(K38A) expressing cells (Fig. 5C), thus demonstrating that both TBK1 and IKK specifically phosphorylate the C-terminal domain of cRel and redirect cRel into the nucleus.

View larger version (38K): [in this window] [in a new window]   FIGURE 5. TBK1 ectopic expression induces cRel phosphorylation and nuclear accumulation. A, HEK 293T cells were transfected with a plasmid encoding Myc-tagged cRel without (lane 1) or with an increasing amount of Flag-tagged TBK1wt (lanes 2–6) encoding plasmid. At 24 h posttransfection, whole cell extracts were prepared and resolved by SDS-PAGE and proteins were revealed by immunoblot using anti-cRel, anti-Flag, or anti-actin Abs. B, Recombinant TBK1 was expressed in baculovirus/insect cell system and purified as described elsewhere (19 ). Recombinant purified TBK1 was used in in vitro kinase assays with purified GST-cRel A, GST-cRel B, and GST-cRel C fusion proteins as substrates. Substrates were stained with Coomassie blue (CB) and 32P incorporation (KA) was measured by autoradiography. C, HeLa cells were cotransfected with plasmids encoding Myc-tagged cRel (1 µg) and either the GFP encoding vector as control (1 µg) or vector encoding GFP-TBK1wt-expressing (1 µg) or GFP-TBK1 K38A-expressing (1 µg) plasmids. After 48 h of expression, cells were fixed and stained with Cy3-labeled anti-Myc (red) and nuclei were revealed with Hoechst 33342 (blue).

IKK-induced cRel nuclear accumulation is independent of IB Ser³² and Ser³⁶ phosphorylation

In a first attempt to determine whether the observed effect on cRel nuclear translocation was a result of IKK-mediated IB Ser³² and Ser³⁶ phosphorylation and degradation (11, 12), Flag-tagged wtIKK was expressed with a 1:1 ratio of Myc-tagged cRel, wtIB, or IB2N (which harbors Ser³²/Ser^36A substitutions and therefore cannot be phosphorylated or degraded upon stimulation and acts as a dominant negative inhibitor of NF-B transactivation (38). As revealed by indirect immunofluorescence (Fig. 6A), cRel was mainly nuclear in IKK-expressing cells in the presence of IB or IB2N, with 94 and 91% of the cells showing exclusively nuclear cRel localization, respectively. Furthermore, immunoblot analysis showed that IB2N was not phosphorylated following IKK expression (Fig. 6B, lane 4), whereas wtIB was clearly phosphorylated as a consequence of IKK expression (Fig. 6B, lane 3). Significantly, the presence of IB2N did not interfere with IKK-induced appearance of the slowly migrating phosphorylated form of cRel (Fig. 6B, lanes 3 and 4).

View larger version (36K): [in this window] [in a new window]   FIGURE 6. IKK-mediated cRel nuclear translocation is independent of IB Ser32 and Ser36 phosphorylation. 293T cells were transfected with plasmids encoding Myc-tagged cRel and Flag-tagged IKKwt in combination with plasmids expressing IB or a dominant negative form of IB (IB2N). A, Cells were fixed and stained with Cy3-labeled anti-Myc, FITC-labeled anti-Flag, and nuclei were revealed with Hoechst 33342. Percentage of cells expressing cRel in the nucleus was determined by counting cells coexpressing Myc-cRel and Flag-IKK. Quantification represents the average of three x40 fields for each condition. B, Whole cell extracts were resolved by SDS-PAGE followed by immunoblot analysis using anti-cRel, anti-Flag, anti-phospho-IB, anti-IB, or anti-actin Abs. Data are representative of three different experiments.

IKK induces dissociation of the IB-cRel complex independent of Ser³² and Ser³⁶

Based on the above result, nuclear and cytoplasmic extracts were prepared to determine whether IB could translocate together with cRel into the nucleus. Flag-tagged wtIKK was coexpressed with a 1:1 ratio of Myc-tagged cRel and Flag-tagged IB2N in HEK 293T cells. As observed with indirect immunofluorescence (Fig. 4), biochemical fractionation confirmed that IKK induced cRel nuclear translocation (Fig. 7, lane 2 and lane 3), whereas IB remained exclusively cytoplasmic (Fig. 7, lane 5 and lane 6 vs lane 2 and lane 3). Next, coimmunoprecipitation was performed to examine whether IB and cRel were physically associated in the cytoplasmic fraction in the presence of IKK. In the absence of IKK, IB2N and cRel coimmunoprecipitated as a cytoplasmic complex (Fig. 7, lane 4), whereas IKK expression disrupted the IB2N-cRel interaction (Fig. 7, lane 6). These results demonstrate that IKK induces cRel nuclear translocation through a mechanism that involves the phosphorylation-dependent dissociation of the cRel-IB2N complex, without degradation of IB.

View larger version (33K): [in this window] [in a new window]   FIGURE 7. IKK induces dissociation of the IB-cRel complex independently of Ser32 and Ser36 phosphorylation. HEK 293T cells were cotransfected with plasmids encoding Flag-tagged IB2N and Myc-tagged cRel in a 1:1 ratio along with an empty vector (lanes 1 and 4) or Flag-tagged IKKwt (lanes 2, 3, 5, and 6). Nuclear and cytoplasmic fractions were prepared as described in Materials and Methods and analyzed by immunoblot (IB). Purity of the fractions was verified through the use of anti-poly(ADP-ribose) polymerase (PARP) and anti-tubulin Abs. Cytoplasmic extracts were coimmunoprecipitated (IP) using an anti-Myc Ab. Immunocomplexes were resolved by SDS-PAGE and analyzed by immunoblot. Quantification of the IB2N to cRel ratio was performed using the ImageJ software. Data are representative of three experiments.

IKK-induced cRel phosphorylation and nuclear accumulation is independent of IKK activity

Both TBK1 and IKK have been considered upstream of IKK in the NF-B activation pathway (45, 46). Thus, the potential involvement of IKK in IKK-mediated cRel nuclear accumulation was analyzed by indirect immunofluorescence following expression of cRel, IB and either IKK or IKK. As expected, IKK expression induced nuclear accumulation of cRel in 98% of cells compared with 2% of control cells (Fig. 8, A and B, lanes 1 and 2). Furthermore, expression of increasing amounts of a dominant negative form of IKK did not inhibit IKK-mediated cRel nuclear accumulation (Fig. 8, A and B, lanes 3 and 4). In contrast, dominant negative form of IKK completely blocked IKK-induced nuclear translocation of cRel (Fig. 8, A and B, lanes 7 and 8). Immunoblot analysis confirmed that cRel was phosphorylated both in the absence or presence of the dominant negative form of IKK (Fig. 8C), thus indicating that IKK expression induces cRel nuclear accumulation independently of IKK activity.

View larger version (39K): [in this window] [in a new window]   FIGURE 8. IKK-mediated cRel nuclear translocation is independent of IKK activation. HEK 293T cells were cotransfected with plasmids encoding IB and Myc-tagged cRel in a 1:1 ratio together with an empty vector (lane 1), Flag-tagged IKKwt (lanes 2–4), or Flag-tagged IKK (lanes 7 and 8), and/or Flag-tagged IKKDN (lanes 3–6). A, Cells were fixed and stained with Cy3-labeled anti-Myc (red), FITC-labeled anti-Flag (green), and nuclei were revealed with Hoechst 33342 (blue). B, Cells from samples in A were counted for cRel nuclear localization. Three x40 fields were counted for each condition and only cells expressing Flag-tagged IKK and IKK (where transfected) were counted. Data represented in the graph correspond to the average of the three fields ± SE. C, Whole cell extracts derived from cells expressing IB and Myc-tagged cRel in a 1:1 ratio along with an empty vector (lane 1), Flag-tagged IKKwt (lanes 2 and 3) and/or Flag-tagged IKKDN (lanes 3 and 4) were resolved by SDS-PAGE and analyzed by immunoblot using anti-cRel, anti-Flag, or anti-actin Abs. Data are representative of three different experiments.

	Discussion

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cRel TD is phosphorylated on Ser⁴⁷¹ in response to TNF- stimulation and this leads to increased cRel transcriptional activity (34, 35). cRel is also phosphorylated and translocated to the nucleus following TCR stimulation by PMA/anti-CD28 (36), where nuclear cRel remains detectable for several days post-TCR stimulation (49). However, in contrast to RelA/p65, where mutation of Ser⁵³⁶ to Ala is sufficient to abrogate IKK and TBK1-induced phosphorylation (24), cRel appears to have multiple sites for IKK-mediated phosphorylation. The use of smaller peptides of cRel TD demonstrated that IKK and TBK1 phosphorylate at least two sites contained within aa 422–472 and aa 532–587. However, attempts to mutate the individual consensus phosphoacceptor sites in the TD did not lead to a significant decrease in IKK-inducible cRel phosphorylation. In contrast, deletion of the TD led to a significant but not complete decrease in the IKK-inducible phosphorylation of cRel (data not shown). Taken together, these results indicate the importance of the TD as a target for IKK-mediated phosphorylation and also suggest the existence of other phosphoacceptor sites in the cRel molecule. Many IKK and TBK1 consensus sequences have been identified within the cRel primary sequence, making it a complex task to identify the combination of sites targeted by these two kinases.

IKK-induced cRel phosphorylation was not sufficient to induce a dramatic increase in cRel transactivation potential (Fig. 4B). The use of a Gal4-cRelTD fusion protein to measure cRel transactivation activity also failed to activate cRel (data not shown); however, this result should be interpreted with caution, as a negative result may reflect the constitutive nuclear localization of the Gal4-cRelTD protein, whereas IKK and TBK1 phosphorylate cRel localized to the cytoplasm, in association with IB. Nuclear retention of phosphorylated cRel was not sufficient to induce strong cRel transactivation, suggesting that an additional modification may be required to stimulate cRel transactivation. This activation step may involve additional cRel posttranslational modification, association with coactivators or chromatin modification.

The current model regarding NF-B sequestration in the cytoplasm by IB involves an equilibrium in the nucleocytoplasmic shuttling of IB-NF-B complexes in favor of cytoplasmic localization, with nuclear NF-B remaining inactive due to its association with IB. Following stimulus-induced IB degradation, the NF-B dimer binds DNA and activates gene transcription (1). A schematic diagram illustrating the role of TBK1 and IKK in cRel activation is shown in Fig. 9. In this model, the IB-cRel complex shuttles between the cytoplasm and nucleus with a balance in favor of cytoplasmic localization in unstimulated cells. IKK-mediated phosphorylation of cRel (concomitant with IB Ser³⁶ phosphorylation) induces a conformational change that causes dissociation of the IB-cRel complex in the cytoplasm and permits nuclear accumulation, independent of IB degradation. As nuclear accumulation does not appear to be sufficient to induce full cRel transactivation, it is likely that another regulatory step is required for full activation. In fact, IKK may play a role in this process. Indeed, we found that cRel was also a substrate for IKK, in the cRel B and cRel C domains of the TD (Fig. 3). Furthermore, in addition to a well-characterized cytoplasmic localization, IKK and IKK are also present in the cell nucleus (50, 51). IKK was shown to modify histones via phosphorylation, a mechanism essential for the activation of NF-B-directed gene expression (50, 51). More recently, it was reported that in primary neutrophils, IKK and RelA/p65 are constitutively localized to the nucleus, whereas TBK1 and IKK remain exclusively cytoplasmic (52). It remains a possibility that a two-step modification of cRel is required for nuclear translocation and full transcriptional activation.

View larger version (19K): [in this window] [in a new window]   FIGURE 9. A model for the role of IKK-mediated phosphorylation in cRel activation. In unstimulated cells, complex containing cRel and IB shuttle from the cytoplasm to the nucleus, with a balance in favor of the cytoplasm. In the cytoplasm, IKK phosphorylates (P) cRel and IB on Ser36, resulting in the dissociation of the IB/cRel containing dimers. The freed cRel accumulates in the nucleus, where it undergoes additional regulation for full transcriptional activity.

In the classical pathway, the IKK-mediated phosphorylation and degradation of IB is transient and newly synthesized IB replenishes the cytoplasmic pool. Interestingly, NF-B activation is also suggested to induce IKK expression, as it is induced following several stimuli that activate NF-B, such as LPS, PMA, TNF-, IFN-, or IL-6 stimulation (12), viral infection, TLR3 and TLR4 stimulation (55) as well as PMA or TCR stimulation (11, 46). IKK may thus be important for the sustained activation of de novo synthesized cRel, and may be important during T cell activation, when cRel is required for sustained IL-2 production (reviewed in Ref. 56).

During the course of this study, Mattioli et al. (57), reported that IKK was involved in the phosphorylation of RelA/p65 at Ser⁴⁶⁸ in response to T cell costimulation and demonstrated that this phosphorylation primarily affected RelA/p65-dependent transactivation. Thus, the function of IKK-mediated phosphorylation of RelA/p65 appears distinct from the function described here for cRel. In conclusion, the present results demonstrate that IKK- and TBK1-mediated phosphorylation of cRel in the C-terminal TD leads to cytoplasmic dissociation of a cRel-IB complex and nuclear accumulation of cRel. Further studies are underway to determine whether a second IKK-mediated phosphorylation event may be required for full cRel activation.

	Acknowledgment

 
We thank members of the Terry Fox Molecular Oncology Group of the Lady Davis Institute for helpful discussions.

	Disclosures

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The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by grants from the National Cancer Institute of Canada (to J. Hiscott) with funds from the Canadian Cancer Society, the Canadian Institutes of Health Research (CIHR), and the Canadian Network for Vaccines and Immunotherapeutics. J. Harris was supported by an Natural Sciences and Engineering Research Council studentship. S.S. was supported by a CIHR studentship. R.L. was supported by a Le Fonds de la Recherche en Santé du Québec (FRSQ) Chercheur Boursier. J. Hiscott is the recipient of the CIHR Senior Scientist Award. N.G. is the recipient of a postdoctoral FRSQ fellowship.

² Current address: Center for Blood Research, Harvard Medical School, Boston, MA 02115.

³ Address correspondence and reprint requests to Dr. John Hiscott, Terry Fox Molecular Oncology Group, Lady Davis Institute for Medical Research, 3755 chemin de la Cote Sainte Catherine, Montréal, Québec H3T 1E2, Canada; or Dr. Nathalie Grandvaux, Centre de Recherche du Centre Hospitalier de l’Université de Montréal, 264 Boulevard René Levesque est, PEA-311, Montréal, Québec, H2X 1P1, Canada; E-mail addresses: john.hiscott{at}mcgill.ca or nathalie.grandvaux{at}umontreal.ca

⁴ Abbreviations used in this paper: TD, transactivation domain; IKK, IB kinase; IRF, IFN regulatory factor; TRAF, TNFR-associated factor; TANK, TRAF family member-associated NF-B activator; TBK, TANK-binding kinase.

Received for publication July 13, 2005. Accepted for publication May 19, 2006.

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