ABSTRACT
Tumor progression locus 2 (TPL-2) functions as a MEK-1/2 kinase, which is essential for Toll-like receptor 4 (TLR4) activation of extracellular signal-regulated kinase 1 and 2 (ERK-1/2) mitogen-activated protein (MAP) kinases in lipopolysaccharide (LPS)-stimulated macrophages and for inducing the production of the proinflammatory cytokines tumor necrosis factor and interleukin-1β. In unstimulated cells, association of TPL-2 with NF-κB1 p105 prevents TPL-2 phosphorylation of MEK-1/2. LPS stimulation of TPL-2 MEK-1/2 kinase activity requires TPL-2 release from p105. This is triggered by IκB kinase 2 (IKK-2) phosphorylation of the p105 PEST region, which promotes p105 ubiquitination and degradation by the proteasome. LPS activation of ERK-1/2 additionally requires transphosphorylation of TPL-2 on serine 400 in its C terminus, which controls TPL-2 signaling to ERK-1/2 independently of p105. However, the identity of the protein kinase responsible for TPL-2 serine 400 phosphorylation remained unknown. In the present study, we show that TPL-2 serine 400 phosphorylation is mediated by IKK2. The IKK complex therefore regulates two of the key regulatory steps required for TPL-2 activation of ERK-1/2, underlining the close linkage of ERK-1/2 MAP kinase activation to upregulation of NF-κB-dependent transcription.
INTRODUCTION
Innate immune responses are triggered by the stimulation of pathogen recognition receptors (PRR) by invariant pathogen molecules, termed pathogen-associated molecular patterns (PAMPs) (19). For example, lipopolysaccharide (LPS) on Gram-negative bacteria binds to Toll-like receptor 4 (TLR4) on macrophages to induce the expression of several hundred genes that together regulate inflammatory and innate immune responses (32). LPS induction of these genes involves several intracellular signaling pathways, including those leading to stimulation of nuclear factor κB (NF-κB) transcription factors and each of the major mitogen-activated protein (MAP) kinase subtypes (extracellular signal-regulated kinases 1 and 2 [ERK-1/2], Jun amino terminal kinases 1, 2, and 3 [JNK-1/2/3], and p38α/β).
The MAP3 kinase tumor progression locus 2 (TPL-2) functions as a MEK-1/2 kinase, which mediates activation of ERK-1/2 MAP kinases by all TLRs in macrophages (10). ERK-1/2 activation by tumor necrosis factor receptor 1 (TNF-R1) and interleukin-1 receptor (IL-1R) is also mediated by TPL-2, consistent with its important role in inflammatory responses (9, 26). Following LPS stimulation of macrophages, TPL-2 promotes the production of TNF, IL-1β, and IL-10 while inhibiting the production of IL-12 and IFN-β (8, 15, 26, 39). TPL-2, therefore, has complex proinflammatory and anti-inflammatory effects on cytokine production in macrophages. Nevertheless, disease model experiments with TPL-2 knockout mice suggest that the net effect of TPL-2 signaling in the innate immune system is to promote inflammation (10). For example, TPL-2 is required for the development of Crohn's-like inflammatory bowel disease that develops in TnfΔARE/ΔARE mice, which constitutively overproduce TNF (21), and in LPS-induced septic shock (8).
In unstimulated cells, all detectable TPL-2 forms a high-affinity stoichiometric complex with NF-κB1 p105, an NF-κB inhibitory protein (IκB) and precursor of the NF-κB p50 subunit (3, 5). Binding to p105 maintains steady-state levels of TPL-2 protein and also negatively regulates TPL-2 activation of ERK-1/2 by preventing access to its substrates MEK-1/2 (3, 37). LPS activation of TPL-2 MEK-1/2 kinase activity requires its release from p105. This is triggered by phosphorylation of p105 by the IκB kinase (IKK) complex, comprised of IKK1 (IKKα), IKK2 (IKKβ), and NEMO, which induces p105 K48-linked ubiquitination and subsequent proteolysis by the proteasome (4, 36). The IKK complex additionally regulates the activation of NF-κB transcription factors through its ability to phosphorylate IκBs, including IκBα and p105, which retain NF-κB dimers in the cytoplasm of unstimulated cells (12, 17). This induces IκB degradation, releasing associated NF-κB to translocate into the nucleus and modulate gene transcription. Activation of NF-κB and ERK-1/2 during an innate immune response is therefore directly coupled via IKK complex phosphorylation of NF-κB1 p105.
TPL-2 is also regulated independently of p105 by phosphorylation, similar to other MAP3 kinases (34). We showed previously that the inducible phosphorylation of serine 400 (S400) in the C-terminal tail of TPL-2 is essential for LPS activation of ERK-1/2 in macrophages (29). Retroviral reconstitution experiments have shown that S400 phosphorylation is required for TPL-2 activation of ERK-1/2 in Nfkb1−/− macrophages. Furthermore, in Nfkb1+/+ cells, S400 phosphorylation is required for LPS activation of ERK-1/2 but is not needed for release of TPL-2 from p105. Therefore, S400 phosphorylation regulates a step in TPL-2 signaling that is distinct from its release from its inhibitor p105. S400 is not autophosphorylated in macrophages (29), but the identity of the TPL-2 S400 kinase has remained unknown.
In the present study, we investigated the possibility that the IKK complex directly phosphorylates TPL-2 to regulate TPL-2-dependent ERK-1/2 activation. Using a combination of pharmacologic, genetic, and biochemical approaches, we showed that IKK2 directly regulates TPL-2 signaling by phosphorylating TPL-2 S400. The IKK complex, therefore, controls two independent steps in the activation of the TPL-2/ERK-1/2 MAP kinase pathway: phosphorylation of the key regulatory residue S400 in the C terminus of TPL-2 and phosphorylation of NF-κB1 p105 to induce p105 proteolysis, releasing TPL-2 from p105-mediated inhibition.
MATERIALS AND METHODS
Mouse strains.Mouse strains were bred in a specific-pathogen-free environment at the National Institute for Medical Research (London, United Kingdom), and all experiments were done in accordance with regulation of the Home Office of the United Kingdom. Map3k8−/−, Nfkb1−/−, Nfkb1SSAA/SSAA, and Nfkb1SSAA/SSAA × Map3k8D270A/D270A mouse strains have been described previously (8, 31, 33, 38) and were fully backcrossed to C57BL/6. Ikk2fl/fl LysM-Cre and control Ikk2fl/fl mice were generated as before (16).
Reagents.Antibodies to TPL-2 (M20; H-7; Santa Cruz) and ERK-1/2 were obtained from Santa Cruz. Antibody to activated phospho-(T185/Y187)-ERK-1/2 (P-ERK) was obtained from Biosource. Antibodies against p38 and phospho-(T180/Y182)-p38 (P-p38) were purchased from Cell Signaling Technology. Phospho-S400 TPL-2, p105, ABIN-2, and IKK2 antibodies have been described previously (22, 23, 29, 30).
The IKK2 inhibitor BI605906 was used at a final concentration of 10 μM (7). BI605906 was dissolved in dimethyl sulfoxide (DMSO) and added 1 h prior to stimulation with LPS. Control cells were treated with an equivalent volume of DMSO vehicle.
In vitro generation and stimulation of macrophages.Bone marrow-derived macrophages (BMDM) were prepared as described previously (35). Briefly, bone marrow cells were plated in complete BMDM medium (RPMI 1640 medium [Sigma] supplemented with 10% heat-inactivated fetal bovine serum [FBS], 20% L-cell conditioned medium, 2 mM glutamine, nonessential amino acids, antibiotics, 10 mM HEPES, and 50 μM β-mercaptoethanol). On day 4 of culture, cells were further supplemented with complete BMDM medium. After a total of 7 days of culture, adherent macrophages were harvested for experiments. Flow cytometric analysis indicated that >95% of these cells were positive for the macrophage markers F4/80 and CD11b. BMDM were replated in Nunc tissue culture dishes (six-well plates, 1 × 106 cells/well; 60-mm dishes, 3 × 106 cells) in BMDM medium containing reduced FBS (1%) and no L-cell conditioned medium. Cells were then cultured overnight, prior to stimulation with LPS (Salmonella enterica serovar Minnesota R595; Alexis Biochemicals) at a final concentration of 10 or 100 ng/ml, unless specified otherwise in the figure legends.
Retrovirus infection of macrophages.Amphoteric recombinant retroviruses were produced by transfecting wild-type (WT) and mutant TPL-2 cDNAs, subcloned in the pMX vector, into the Plat-E packaging cell line (27), using the GeneJuice transfection reagent (Merck Biosciences). Transfected cells (3 × 106 cells/well of a P90 plate; Nunc) were cultured at 37°C for 24 h in complete medium (Dulbecco's modified Eagle medium [DMEM] plus 10% FBS and antibiotics). Culture medium was replaced, and virus-containing supernatant was harvested at 24 h, filtered (pore size, 0.4 μm), and concentrated by spinning at 20,000 rpm for 3 h at 4°C.
For retroviral infection of Map3k8−/− and Nfkb1−/− BMDM, BM cells were plated in complete BMDM medium at 1 × 106 cells/well of six-well plate (2 ml culture volume; Sarstedt). Following 4d of culture, 200 μl of virus containing supernatant was added per well, and plates were centrifuged at 2,000 × g for 1 h. Cells were cultured for 3 h, 4 ml complete BMDM medium was added, and cells were recultured for a further 4 days. Cell harvested at this time were >95% F4/80+. For experiments, cell were replated at 1 × 106 cells/well (2 ml culture volume) of a six-well plate (Nunc) in RPMI medium plus 1% FBS.
293 cell culture and transfection.QBI-293A cells were plated at 2 × 105 cells/well (1 ml culture volume) of a six-well plate (Nunc) in DMEM plus 10% FBS. After 24 h, medium was replaced with DMEM with no added FBS, and cells were cultured overnight, prior to stimulation with TNF (20 ng/ml; R & D Systems) for 15 min.
HEK-293 cells stably expressing the IL-1R (C6 cells) were kindly provided by Xiaoxia Li (The Cleveland Clinic Foundation, Cleveland, OH) (24). QBI-293A and C6 cells were transiently transfected using Lipofectamine and Lipofectamine 2000 (Life Technologies Inc.), respectively. Cells were cultured for a total of 48 h, as described previously (3). C6 cells were stimulated with recombinant IL-1β (Peprotech), used at a final concentration of 20 ng/ml.
Protein analyses.Cells were washed once in phosphate-buffered saline (PBS) before lysis. For immunoblots of total lysates, cells were lysed in buffer A (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 50 mM NaF, 1 mM Na3VO4, 100 nM okadaic acid [Calbiochem], 2 mM Na4P2O7 plus protease inhibitors [Roche Molecular Biochemicals]) containing 1% Nonidet-P40. After centrifugation to remove particulate matter, 20 to 40 μg of protein lysate was mixed with an equal volume of 2× sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer and then immunoblotted. For immunoprecipitations, lysis was carried out using buffer A containing 0.5% Nonidet-P40 and 1 mM dithiothreitol (DTT) (1 to 2 mg of protein lysate per immunoprecipitation). Myc-TPL-2 and FLAG–TPL-2 were immunoprecipitated using c-Myc monoclonal antibody (MAb) agarose affinity gel (Sigma) and M2 FLAG MAb agarose affinity gel (Sigma), respectively.
Production of recombinant TPL-2/NF-κB1 p105/ABIN-2 complex.Recombinant TPL-2D270A/NF-κB1 p105/ABIN-2 complex was purified from QBI-293A cells transiently transfected with expression vectors encoding human His6-TPL-2D270A, ABIN-2-StrepII, and HA-p105. At 72 h after transfection, cells were lysed in 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.5% NP-40, 0.5 mM Tris(2-carboxyethyl)phosphine (TCEP), 10% glycerol, and 10 mM imidazole (supplemented with protease and phosphatase inhibitors). Lysates were subjected to three-step affinity purification using nickel-nitrilotriacetic acid (Ni-NTA) agarose (Qiagen), StrepTactin Sepharose (GE Healthcare), and 1 ml HisTrap HP columns (GE Healthcare). Purified protein was eluted in 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1.8 mM decyl-β-d-maltopyranoside, 0.5 mM TCEP, 10% glycerol, and 200 mM imidazole. Purity was estimated to be 91.5%, as determined by measurement of the infrared fluorescence of Coomassie blue-stained SDS-PAGE gels. A detailed description of this method will be reported in a separate publication.
In vitro phosphorylation of recombinant TPL-2 complex by IKK2.Purified His6-TPL-2D270A/HA-p105/ABIN-2-StrepII complex (250 nM) was incubated with recombinant His6-IKK2 (50 nM; gift from P. Cohen, Dundee, United Kingdom) in 20 mM Tris (pH 8.0), 150 mM NaCl, 0.03% Brij-35, 5 mM β-glycerophosphate, and 2 mM DTT with or without 100 μM BI605906 in the presence of 10 mM MgCl2, 0.1 mM ATP, and 0.01 μCi/μl [γ-32P]ATP for 30 min at 30°C. Kinase reactions were stopped by boiling in Laemmli sample buffer, and then the products were resolved by SDS-PAGE. Phosphorylated proteins were detected by autoradiography of the dried gel. For mass-spectrometric analyses, purified His6-TPL-2D270A/HA-p105/ABIN-2-StrepII complex was incubated with or without His6-IKK2 as described above but without addition of [γ-32P]ATP. Incubation times were also prolonged to 4 h to increase the stoichiometry of phosphorylation.
Mass-spectrometric analysis of TPL-2 phosphorylation sites.In vitro-phosphorylated TPL-2D270A/ABIN-2/NF-κB1 p105 complex was boiled in 1% SDS with 10 mM DTT and concentrated by trichloroacetic acid precipitation and centrifugation. Precipitated protein was dissolved in Laemmli sample buffer supplemented with 10 mM DTT, incubated with 50 mM iodoacetamide to alkylate cysteine residues, and then resolved by SDS-PAGE (10% Novex NuPAGE gel; Life Technologies Inc.). Proteins were visualized by colloidal Coomassie blue staining, and bands were excised in 2-mm cubes. After washing with 100 mM triethylammonium bicarbonate (TEAB) (pH 8.5) and 50% acetonitrile–100 mM TEAB, dried gel pieces were digested with 2.5 μg/ml trypsin. Peptides were analyzed by liquid chromatography-mass spectrometry (LC-MS) on an LTQ-Orbitrap Velos mass spectrometer system, coupled to a Proxeon Easy-LC high-pressure liquid chromatograph (HPLC). Tandem MS (MS/MS) spectra were searched against the Swiss-Prot database using the Mascot search engine (Matrix Science). Qual Browser software was used to analyze all MS/MS spectra assigned to a phosphopeptide manually.
RESULTS
IKK2 regulates TPL-2 phosphorylation in LPS-stimulated macrophages.In an attempt to identify TPL-2 regulatory kinases, we screened a commercial small-interfering-RNA (siRNA) library (Ambion), which targets each kinase in the human genome with three different siRNA oligonucleotides. Since available phospho-S400 antibodies are very insensitive, we assayed TNF induction of ERK-1/2 phosphorylation, which was mediated via TPL-2 in the QBI-293A target cells used. The screen identified several kinases that were already known to be required for TNF activation of ERK-1/2 (10, 18), including TAK1 (TAB2 identified), IKK1/IKK2/NEMO, TPL-2, and MEK-1/2, confirming that the assay was working. Other kinases were identified in this initial screen, which appeared to be required for optimal activation of ERK-1/2 by TNF. However, after further extensive testing (by deconvolution of siRNA oligonucleotide pools and use of additional siRNAs binding to different regions of the targeted genes), none of these could be validated.
Since it was not possible to check whether each kinase targeted by the siRNA library was efficiently knocked down, the siRNA screen may have failed to detect novel regulatory kinases of the TPL-2/ERK-1/2 pathway for technical reasons. However, it was also possible that TPL-2 phosphorylation on critical regulatory residues was mediated by a kinase already known to be important in TNF activation of ERK-1/2. Earlier experiments had demonstrated that IKK-induced phosphorylation and proteolysis of p105 required IKK association with the p105 death domain (2). TPL-2 also interacts with the p105 death domain (3), which could bring IKK and TPL-2 into close proximity. Furthermore, it has been shown that TPL-2 can directly interact with IKK (25). We therefore investigated whether IKK might regulate TPL-2 signaling via direct phosphorylation, in addition to inducing the proteolysis of NF-κB1 p105 (4).
TPL-2 is expressed in two forms, M1-TPL-2 and M30-TPL-2, due to translational initiation on alternative methionines (at residues 1 and 30) (1). LPS stimulation of macrophages induces a clear mobility shift of M1-TPL-2 on SDS-PAGE, which is due to phosphorylation and is followed by its degradation by the proteasome (4). M30-TPL-2 is also phosphorylated at a lower stoichiometry and is consequently more difficult to visualize after SDS-PAGE. In initial experiments, the effect of pharmacological inhibition of IKK2 was tested on LPS-induced M1-TPL-2 phosphorylation. Pretreatment of murine bone marrow-derived macrophages (BMDM) with the highly selective IKK2 inhibitor BI605906 (7) blocked LPS from inducing both M1-TPL-2 phosphorylation and subsequent proteolysis (Fig. 1A). BI605906 inhibited LPS-induced phosphorylation of ERK-1/2, as expected. Consistent with these results, stimulation of QBI-293A cells with TNF induced a mobility shift in M1-TPL-2 that was also blocked by BI605906 (Fig. 1B).
IKK2 regulates LPS-induced phosphorylation of TPL-2. Cell lysates were immunoblotted for the antigens shown. (A, D, and E) BMDM of the indicated genotypes were pretreated with BI605906 or vehicle control (DMSO) and then stimulated with LPS (10 ng/ml) or left unstimulated. WT cells were used in panel A. In panels D and E, no ERK-1/2 phosphorylation was detected after LPS stimulation with or without BI605906 (data not shown). (B) QBI-293A cells were pretreated with BI605906 or vehicle control (DMSO) and then stimulated with TNF for 15 min or left unstimulated. (C) BMDM of the indicated genotypes were stimulated with LPS or left unstimulated.
Since pharmacological inhibitors can have off-target effects, it was important to determine genetically whether IKK2 regulated M1-TPL-2 phosphorylation. To do this, BMDM were cultured from Ikk2fl/fl LysM-Cre mice, in which IKK2 is specifically deleted in myeloid cells (16). IKK2 expression was only partially decreased in Ikk2fl/fl LysM-Cre BMDM compared to that in Ikk2fl/fl controls. Consequently, LPS-induced phosphorylation of p105 and ERK-1/2 in Ikk2fl/fl LysM-Cre BMDM was decreased relative to that in Ikk2fl/fl controls but was not blocked (Fig. 1C). Nevertheless, the LPS-induced mobility shift of M1-TPL-2 was clearly reduced (Fig. 1C), confirming that IKK2 does regulate TPL-2 phosphorylation.
IKK2 directly phosphorylates two serines in the PEST region of NF-κB1 p105 (serines 930 and 935 in murine p105) to induce its proteolysis by the proteasome (3, 5, 22). To investigate whether IKK2 regulation of M1-TPL-2 phosphorylation was mediated via phosphorylation of NF-κB1 p105, the effect of BI605906 was tested on BMDM generated from Nfkb1SSAA/SSAA mice, in which the IKK phosphorylation sites on p105 are mutated to alanines. We have shown previously that Nfkb1SSAA mutation blocks LPS-induced release from p105, abrogating TPL-2 activation of ERK-1/2 (38). The Nfkb1SSAA mutation also prevented M1-TPL-2 proteolysis but did not affect the phosphorylation shift of M1-TPL-2 after LPS stimulation (Fig. 1D). However, BI605906 blocked LPS-induced M1-TPL-2 phosphorylation in Nfkb1SSAA/SSAA BMDM. IKK2, therefore, regulated M1-TPL-2 phosphorylation independently of p105 phosphorylation and the release of TPL-2 from p105.
Previous results suggested that LPS stimulation induces TPL-2 autophosphorylation in macrophages (29). It was therefore possible that IKK2 regulates M1-TPL-2 phosphorylation indirectly by inducing TPL-2 autophosphorylation. To investigate this possibility, BMDM were generated from Nfkb1SSAA/SSAA Map3k8D270A/D270A mice, which express catalytically inactive TPL-2. The effect of TPL-2 kinase activity was tested in an Nfkb1SSAA/SSAA background in which M1-TPL-2 proteolysis was blocked (Fig. 1E), and consequently M1-TPL-2 phosphorylation was easier to detect. LPS stimulation induced a clear phosphorylation mobility shift in TPL-2D270A (Fig. 1E). BI605906 completely blocked this mobility shift, which was confirmed to be due to phosphorylation by phosphatase treatment of immunoprecipitated TPL-2D270A (data not shown).
Together, the results in this section indicated that LPS stimulated M1-TPL-2 transphosphorylation in macrophages, and this was dependent on IKK2 catalytic activity. TNF-induced M1-TPL-2 phosphorylation also required IKK2 catalytic activity, suggesting a general role for IKK2 in regulating TPL-2 phosphorylation.
IKK2 directly phosphorylates TPL-2 in vitro.The previous experiments raised the possibility that IKK2 directly phosphorylated TPL-2 following LPS stimulation, which was investigated with an in vitro kinase assay. Within cells, endogenous TPL-2 is complexed with p105 and the ubiquitin-binding protein ABIN-2, which are both required to maintain TPL-2 protein stability (3, 28, 37). Because IKK2 also regulated the phosphorylation of TPL-2 while still bound to p105, and since it is not possible to produce soluble recombinant TPL-2 protein on its own (14), we developed a method to produce the TPL-2/p105/ABIN-2 complex. To do this, QBI-293A cells were cotransfected with plasmids encoding His6-tagged TPL-2D270A, HA-p105, and ABIN-2-StepII. The TPL-2 complex was then purified by sequential affinity purification via the His6 and StrepII epitope tags, using Ni-NTA agarose and StrepTactin agarose beads, respectively. Mass spectrometry confirmed that the major Coomassie blue-stained bands after SDS-PAGE corresponded to His6-TPL-2D270A, HA-p105, and ABIN-2-StrepII (Fig. 2A). The purity of the isolated TPL-2 complex was estimated to be 91.5%.
Phosphorylation of TPL-2 by IKK2 in vitro. (A) Coomassie blue-stained SDS-PAGE gel of purified TPL-2D270A/NF-κB1 p105/ABIN-2 complex. (B) TPL-2D270A/NF-κB1 p105/ABIN-2 complex was incubated with recombinant IKK2 plus [32P]ATP for a kinase assay (KA). Labeled proteins were revealed by autoradiography after SDS-PAGE. Cell lysates were immunoblotted (IB) for the indicated antigens. (C) Extracted ion chromatograms of phosphorylated S62, S62/S66, and S400 peptides identified by mass spectrometry of TPL-2D270A complex without (left) and with (right) IKK2 phosphorylation. Absolute intensities and retention times are shown (Table 1).
The purified TPL-2 complex was incubated with recombinant IKK2 in the presence of [32P]ATP and then resolved by SDS-PAGE. Autoradiography revealed 32P-labeled bands comigrating with p105, TPL-2, and IKK2 (Fig. 2B), suggesting that IKK2 autophosphorylated and also that it transphosphorylated both TPL-2 and p105. Mass spectrometry revealed that IKK2 phosphorylated TPL-2 on S400, S62, and S66 (Fig. 2C and Table 1) (sequence coverage > 76%). No peptides corresponding to phosphorylated T290 were detected, although IKK2 was suggested to phosphorylate this residue on TPL-2 previously (6), and unphosphorylated peptides containing T290 were evident. These results demonstrated that IKK2 directly phosphorylated TPL-2 on three serines, including the critical regulatory site, S400.
IKK2 phosphorylation sites on TPL-2 indentified by MS
TPL-2 phosphorylation on S62 and S66 is not required for LPS activation of ERK-1/2 in macrophages.Based on overexpression experiments in IL-1R expressing HEK-293 cells, it has been suggested that S62 phosphorylation may regulate the efficiency of TPL-2 signaling to ERK-1/2 (11). However, the significance of S62 and S66 phosphorylation for TPL-2 activation of ERK-1/2 in macrophages was not clear. To investigate this, Map3k8−/− BMDM, which do not express TPL-2 (8), were transduced with recombinant retroviruses encoding wild-type (WT) TPL-2, TPL-2S400A, TPL-2S62A, or TPL-2S66A. LPS stimulation induced levels of ERK-1/2 phosphorylation in cells expressing TPL-2S62A and TPL-2S66A that were comparable to those seen with the WT TPL-2 control (Fig. 3). However, LPS failed to induce ERK-1/2 phosphorylation in BMDM expressing TPL-2S400A, as reported previously (29). TPL-2 S62 and S66 phosphorylation by IKK2, therefore, was not required for TPL-2 activation of ERK-1/2 in macrophages.
TPL-2 S62 and S66 are not required for TPL-2 activation of ERK. Map3k8−/− BMDM were transduced with recombinant retroviruses (RV) encoding TPL-2 (WT), TPL-2S62A, TPL-2S66A, and TPL-2S400A. Cells were stimulated with LPS (100 ng/ml), and lysates were immunoblotted. P-ERK, phospho-ERK-1/2; P-p38, phospho-p38.
IKK2 catalytic activity is required for TPL-2 S400 phosphorylation in cells.Since only S400 was essential for TPL-2 activation of ERK-1/2, we next determined whether IKK2 could regulate phosphorylation of this residue in cells. Overexpression of IKK2 was found to induce S400 phosphorylation of TPL-2 cotransfected with p105 and ABIN-2 (Fig. 4A) or expressed alone (Fig. 4B). P-ERK, phospho-ERK-1/2; T-ERK, total ERK-1/2.
Phosphorylation of TPL-2 S400 by IKK2 in cells. (A) FLAG–TPL-2/HA-p105/ABIN-2-StrepII complex was isolated by anti-FLAG immunoprecipitation (IP) from transiently transfected QBI-293A cells coexpressing HA-IKK2 (+) or empty vector (−). Isolated proteins were resolved by SDS-PAGE and immunoblotted. WCL, whole-cell lysate. (B) C6 cells were transiently transfected with a vector encoding Myc-TPL-2 together with either a vector encoding IKK2 or empty vector (EV). After 48 h, cells were stimulated with IL-1β (20 ng/ml) for 20 min, or left unstimulated. Myc-TPL-2 was immunoprecipitated from cell lysates with Myc MAb and then immunoblotted. (C) C6 cells were transfected with a vector encoding FLAG–TPL-2 or EV. After 48 h, cells were pretreated with BI605906 (+) or vehicle control (−) and then stimulated with IL-1β for 20 min or left unstimulated. FLAG immunoprecipitates were immunoblotted. (D) Nfkb1−/− BMDM, transduced with retrovirus expressing wild-type TPL-2, were pretreated with BI605906 (+) or vehicle control (−) and then stimulated with LPS (100 ng/ml) or left unstimulated. Cell lysates were immunoblotted. P-ERK, phospho-ERK-1/2; T-ERK, total ERK-1/2.
We next investigated whether endogenous IKK2 could phosphorylate TPL-2 S400. Since existing S400 phosphoantibodies are insufficiently sensitive to detect endogenous phosphorylated TPL-2, IL-1R-expressing HEK-293 (C6) cells were transfected with an expression vector encoding FLAG–TPL-2. Stimulation of these cells with IL-1β induced FLAG–TPL-2 S400 phosphorylation (Fig. 4C). Pretreatment of cells with BI605906 prevented IL-1β from inducing FLAG–TPL-2 S400 phosphorylation. Therefore, IL-1β-induced TPL-2 S400 phosphorylation was dependent on endogenous IKK2 activity.
Collectively, our results indicated that IKK2 directly phosphorylated TPL-2 to promote agonist activation of ERK-1/2 in macrophages, and this was independent of IKK phosphorylation of NF-κB1 p105. These results predicted that IKK2 activity would be required for TPL-2-dependent activation of ERK-1/2 in the absence of p105. To investigate this hypothesis, BMDM generated from Nfkb1−/− mice were transduced with recombinant retrovirus encoding TPL-2 to increase the steady-state concentration of TPL-2 in the absence of p105. As shown previously (29), this did not alter basal levels of ERK-1/2 phosphorylation (Fig. 4D), while LPS-induced ERK-1/2 phosphorylation was substantially increased relative to cells transduced with empty vector. However, pretreatment of TPL-2-transduced cells with BI605906 blocked LPS induction of ERK-1/2. This result demonstrated that IKK2 catalytic activity regulated TPL-2-mediated activation of ERK-1/2 in macrophages independently of NF-κB1 p105, consistent with the direct phosphorylation of TPL-2 by IKK2 on the critical regulatory site S400.
DISCUSSION
We provide pharmacological, genetic, and biochemical evidence to demonstrate that IKK2 regulates TPL-2 phosphorylation following LPS stimulation of macrophages. Importantly, we show that IKK2 directly phosphorylates TPL-2 on S400, a residue that was previously shown to be critical for TPL-2-mediated activation of ERK-1/2 in LPS-stimulated macrophages (29). Consistent with this, analysis of the linear sequence surrounding S400 using the known IKK2 consensus motif (13) revealed a similar sequence match of TPL-2 S400 to previously described IKK2 target sites, including IκBα and NF-κB1 p105 (Table 2).
Scansite scores for IKK2 substrates
Our work demonstrates that the IKK complex controls two of the known regulatory steps required for TPL-2 activation of the ERK-1/2 MAP kinase pathway, namely, phosphorylation of NF-κB1 p105 to trigger its proteolysis by the proteasome and direct phosphorylation of the TPL-2 C terminus on S400 (Fig. 5). The significance of linking TPL-2 activation of ERK-1/2 to NF-κB activation via IKK is largely unknown. With respect to the proinflammatory cytokine IL-12, IKK activation of TPL-2/ERK-1/2 signaling has been shown to induce a negative feedback loop to switch off NF-κB-dependent transcription of the Il12a and Il12b genes (38). The regulation of TPL-2 activation by IKK, therefore, limits the proinflammatory effects of NF-κB-induced IL-12. It is, however, unclear whether TPL-2 has a general role in downregulating IKK/NF-κB-dependent gene expression in macrophages. A genome-wide analysis of TPL-2-regulated genes will be necessary to answer this question.
Regulation of TPL-2 activation by IKK. In unstimulated cells, TPL-2 is complexed with NF-κB1 p105. Agonist stimulation induces IKK2, a catalytic subunit of the IKK complex, to phosphorylate p105 on serines 927 and 932 (human p105 numbering) in its PEST region, which triggers p105 K48-linked polyubiquitination and degradation by the proteasome. This releases TPL-2 from p105-mediated inhibition, allowing TPL-2 access to its substrates MEK-1/2. TPL-2 activation of MEK-1/2 also requires phosphorylation of the TPL-2 C terminus on S400, which is shown here to be mediated by IKK2. The IKK complex therefore regulates two critical steps in the activation of TPL-2 MEK-1/2 kinase activity.
ACKNOWLEDGMENTS
We thank P. Tsichlis (Tufts University, Boston, MA) and Thomas Jefferson University for the Map3k8−/− mice, Philip Cohen for IKK2 inhibitor (University of Dundee, Scotland), Mike Howell and Becky Sanders (High Throughput Screening Laboratory, Cancer Research UK, London, United Kingdom) for their help with the siRNA screen, Manolis Pasparakis and Claudia Uthoff-Hachenberg (Institute for Genetics, University of Cologne) for providing BM cells from Ikk2fl/fl LysM-Cre and Ikk2fl/fl mice, the NIMR Photographics Department, NIMR Biological Services, and other members of the Ley laboratory for help during the course of this work.
This work was supported by the U.K. Medical Research Council (U117584209), Arthritis Research UK (grant reference 18864), and Leukemia and Lymphoma Research (grant reference 06050).
FOOTNOTES
- Received 3 August 2012.
- Returned for modification 23 August 2012.
- Accepted 10 September 2012.
- Accepted manuscript posted online 17 September 2012.
- Copyright © 2012, American Society for Microbiology. All Rights Reserved.
