The protein kinase Tpl2 (tumour progression locus 2) is activated by LPS (lipopolysaccharide), TNFα (tumour necrosis factor α) and IL (interleukin)-1. Activation of the native Tpl2 complex by these agonists requires the IKKβ {IκB [inhibitor of NF-κB (nuclear factor κB)] kinase β}-catalysed phosphorylation of the p105/NF-κB1 subunit and is accompanied by the release of the catalytic subunit from both p105/NF-κB1 and another subunit ABIN2 (A20-binding inhibitor of NF-κB 2). In the present study we report that IL-1 activates the transfected Tpl2 catalytic subunit in an HEK (human embryonic kidney)-293 cell line that stably expresses the IL-1R (IL-1 receptor), but does not express the protein kinase IRAK1 (IL-1R-associated kinase). In these cells IL-1 does not activate IKKβ or induce the phosphorylation of p105/NF-κB1, and nor does the IKKβ inhibitor PS1145 prevent the IL-1-induced activation of transfected Tpl2. However, the IL-1-stimulated activation of transfected Tpl2 in IRAK1-null cells or activation of the endogenous Tpl2 complex in IRAK1-expressing cells is suppressed by the protein kinase inhibitor PP2 by a mechanism that does not involve inhibition of Src family protein tyrosine kinases. The IL-1-stimulated activation of transfected Tpl2 is accompanied by its phosphorylation at Thr290 and Ser400 and by enhanced phosphorylation of Ser62, which we demonstrate are autophosphorylation events catalysed by Tpl2 itself. We further show that IL-1 triggers the dissociation of Tpl2 from co-transfected ABIN2 in IRAK1-null IL-1R cells, which is not suppressed by PP2 or by the inhibition of Tpl2 or IKKβ. These studies identify two new signalling events involved in activation of the native Tpl2 complex by IL-1. First, the IRAK1-, IKKβ- and PP2-independent dissociation of Tpl2 from ABIN2; secondly, the IRAK1- and IKKβ-independent, but PP2-sensitive, activation of the Tpl2 catalytic subunit.

INTRODUCTION

The protein kinase Tpl2 (tumour progression locus 2), also called COT (Cancer Osaka Thyroid), plays a pivotal role in the production of TNFα (tumour necrosis factor α). Thus Tpl2-deficient mice fail to secrete TNFα in response to bacterial LPS (lipopolysaccharide) [1], and are protected against LPS-stimulated septic shock and TNFα-induced inflammatory bowel disease [2]. Tpl2 mediates the production of TNFα by activating MKK1 and MKK2 [MAPK (mitogen-activated protein kinase) kinases 1 and 2], which then activate ERK1 and 2 (extracellular-signal-regulated kinases 1 and 2) [1]. ERK1/ERK2 are required for the processing of pre-TNFα to the secreted form of this pro-inflammatory cytokine [3] and contribute to the production of other inflammatory mediators [4,5].

Tpl2 is not only activated by LPS, but also by TNFα [6,7] and IL-1 (interleukin-1) [8], so that pharmacological inhibition of this enzyme should suppress some of the actions of these cytokines, as well as their production. For these reasons, Tpl2 is an attractive target for the development of orally active drugs to treat chronic inflammatory diseases. Indeed, relatively specific pharmacological inhibitors of Tpl2 have been developed that suppress the LPS-stimulated production of TNFα in human monocytes and block inflammatory responses in synoviocytes and blood [9].

The native Tpl2 complex comprises the catalytic subunit and two other proteins, termed p105 [also called NF-κB1 (nuclear factor κB 1)] and ABIN2 (A20-binding inhibitor of NF-κB 2) [10]. The activation of Tpl2 by LPS [7,11] or TNFα [7] requires the phosphorylation of p105 by IKKβ [IκB (inhibitor of NF-κB) kinase β] in primary mouse macrophages or MEFs (mouse embryonic fibroblasts), as phosphorylation was greatly reduced in IKKβ-deficient MEFs and suppressed by PS1145, an IKKβ inhibitor [7,10]. The activation of Tpl2 in macrophages was accompanied by the ubiquitin-mediated proteolysis of p105, and the release of the Tpl2 catalytic subunit from both p105 and ABIN2. The Tpl2 catalytic subunit is expressed in cells as both a ‘long’ form (Tpl2L; amino acid residues 1–467) and a ‘short’ form (Tpl2S; amino acid residues 30–467), which result from the use of alternative initiating methionine residues at residues 1 and 30 [12]. The LPS-stimulated activation of Tpl2 that occurs within minutes correlates with the activation of Tpl2L, which is then also rapidly degraded by the proteasome [7,13].

The presence of both p105 and ABIN2 is essential for Tpl2 stability, and the level of expression of the Tpl2 catalytic subunit is extremely low in either p105−/− [11] or ABIN2−/− [14] primary mouse macrophages. However, when re-transfected into p105−/− macrophages the Tpl2 catalytic subunit was not constitutively active and became active only after stimulation with LPS [15]. This experiment indicated that the activation of Tpl2 requires a second LPS-regulated step that is independent of the phosphorylation of p105 and its dissociation from Tpl2.

The Tpl2 catalytic subunit was reported to undergo phosphorylation at Thr290 when transfected into HEK (human embryonic kidney)-293 cells, and the mutation of this residue to alanine prevented the LPS-stimulated activation of ERK1/ERK2 in transfected mouse RAW macrophages. These experiments suggested that the phosphorylation of Thr290 might be required for the activation of Tpl2 [16]. In addition, a catalytically inactive mutant of Tpl2 (Tpl2[K167M]) was reported to become phosphorylated at Thr290 in transfected HEK-293 cells, suggesting that Thr290 phosphorylation did not occur as a result of autophosphorylation [16,17]. The phosphorylation of Tpl2 at Thr290 was initially reported to be catalysed by IKKβ, based on siRNA (small interfering RNA)-knockdown studies and the use of high concentrations of the IKKβ inhibitor PS1145 [16]. However, subsequent work showed that lower concentrations of PS1145, but nevertheless sufficient to completely inhibit IKKβ, did not affect the IL-1-stimulated phosphorylation of transfected Tpl2 at Thr290 [18]. Thus the IL-1stimulated phosphorylation of Thr290 is catalysed by a protein kinase distinct from IKKβ.

LPS was reported to stimulate the phosphorylation of the endogenous Tpl2 catalytic subunit at Ser400 in primary mouse macrophages. LPS also stimulated Ser400 phosphorylation in an RAW mouse macrophage cell line that had been stably transfected with either wild-type Tpl2 or a catalytically inactive mutant Tpl2[D270A][15]. The phosphorylation of Ser400 was not impaired by BMS-345541 or IKK2 inhibitor IV, two different pharmacological inhibitors of IKKβ. Taken together, these experiments suggested that the phosphorylation of Ser400 was not an autophosphorylation event in these cells and that it was catalysed by a protein kinase distinct from IKKβ. Unlike wild-type Tpl2, the Tpl2[S400A] mutant failed to reconstitute the LPS-stimulated activation of MKK1 and ERK1/ERK2 when transfected into Tpl2−/− or p105−/− macrophages. These experiments suggested that Ser400 phosphorylation is required for the LPS-stimulated activation of the reconstituted Tpl2 complex in Tpl2−/− macrophages and the LPS-stimulated activation of the Tpl2 catalytic subunit in p105−/− macrophages [15]. However, a truncated form of Tpl2 (Tpl2T), comprising amino acid residues 30–397, can be activated by IL-1 in transfected IL-1R (IL-1 receptor) cells [18], indicating that IL-1 can activate Tpl2T by a mechanism that is independent of the phosphorylation of Ser400. The sequence of Tpl2T starts at the same residue as Tpl2S but, like the oncogenic form originally identified in a human thyroid carcinoma [19], it lacks the C-terminal 70 residues. Tpl2T displays enhanced expression compared with Tpl2L and Tpl2S [20].

In the present study, we demonstrate that IL-1 activates the transfected Tpl2 catalytic subunit and induces its dissociation from co-transfected ABIN2 by mechanisms that are independent of IRAK1 (IL-1R-associated kinase) and IKKβ. The activation of the transfected Tpl2 catalytic subunit, but not its dissociation from ABIN2, is suppressed by the protein kinase inhibitor PP2. These findings identify two further signalling events required for the activation of the Tpl2 complex by IL-1.

EXPERIMENTAL

Human IL-1β was expressed as a GST (glutathione transferase)-fusion protein in Escherichia coli, purified on glutathione–Sepharose, cleaved with PreScission proteinase to release IL-1β [117–268], and purified by gel-filtration on Superdex G200 by Dr Gursant Kular in the MRC Protein Phosphorylation Unit. A vector expressing GST–phage λ phosphatase was transformed into E. coli (strain BL21s), the bacteria grown at 37°C until the attenuance at 600 nm was 0.4–0.6 and the expression of the phosphatase was then induced by incubation for 20 h at 26°C with 10 μM IPTG (isopropyl β-D-thiogalactoside). The bacteria were harvested and lysed, and the phosphatase purified on glutathione–Sepharose.

The Src family protein kinase inhibitor PP2, the IKKβ inhibitor PS1145 and staurosporine were purchased from Calbiochem, while the Tpl2 inhibitor called C-1 (Compound-1) [9] and SU6656 [21] were synthesized by Dr Natalia Shpiro in the MRC Protein Phosphorylation Unit.

Antibodies

The peptides DERSKS*LLLS, KDLRGT*EIYMS and DQPRCQS*LDSAL (where S* and T* are phosphoserine and phosphothreonine respectively), corresponding to amino acid residues 57–67, 285–295 and 394–405 of human Tpl2, were synthesized by Dr Graham Bloomberg (University of Bristol, Bristol, U.K.), coupled to both BSA and keyhole limpet haemocyanin and injected into sheep at Diagnostics Scotland. The antibodies were affinity purified on CH–Sepharose to which the relevant phosphorylated peptide had been coupled covalently. The three phospho-specific antibodies recognizing Tpl2 phosphorylated at Ser62 (S705B, bleed 2), Thr290 (S687B, bleed 2) and Ser400 (S027C, bleed 2) were used for immunoblotting at 1 μg/ml. Phospho-specific antibodies were incubated for 1 h with the unphosphorylated form of the peptide immunogen (10 μg/ml) to neutralize any antibodies that might recognize unphosphorylated Tpl2. A further antibody (S219C, bleed 2) that immunoprecipitates Tpl2 was made by injecting purified GST–Tpl2[30–397] (expressed in E. coli) into sheep. These antibodies were purified by affinity chromatography on GST–Tpl2–Sepharose and then freed from anti-GST antibodies by passage through GST–Sepharose. Antibodies that recognize the phosphorylated forms of IKKα/β, IκBα, p105, ERK1/ERK2, p38α MAPK and p130CAS, or which recognize the unphosphorylated and phosphorylated forms of these proteins equally well, were from Cell Signaling Technologies; antibodies that recognize the phosphorylated forms of JNK1/JNK2 (c-Jun N-terminal kinase 1 and 2) were from Biosource; and an antibody that recognizes IRAK1 was from Santa Cruz Biotechnology. Anti-FLAG and anti-HA (haemagglutinin) antibodies for immunoblotting and anti-FLAG–agarose and anti-HA–agarose for immunoprecipitation were from Sigma, whereas rabbit-, mouse- and sheep-specific secondary antibodies conjugated to horseradish peroxidase were from Pierce.

DNA cloning and mutagenesis

DNA encoding Tpl2T (amino acid residues 30–397; GenBank® accession number AY309013) was amplified from IMAGE EST 199903 (http://www.geneservice.co.uk) using the GC-rich PCR system (Roche). The resulting fragment was ligated into pCR2.1 (Invitrogen) and sequenced. pCR2.1-Tpl2[30–397] was digested with BamH1/Not1 and ligated into the same sites in pCMV FLAG2 to produce pCMV FLAG2-Tpl2[30–397] for expression of the FLAG-fusion protein in mammalian cells. Vectors encoding FLAG-tagged Tpl2L (amino acid residues 1–467) and FLAG-tagged Tpl2S (amino acid residues 30–467) were produced in a similar manner. ABIN2 (GenBank® accession number NM_024309) was amplified from IMAGE EST 3632736 as described above and cloned into the BamH1/Not1 sites of pCMV HA to produced HA-tagged ABIN2 for expression in mammalian cells. Human IL-1β was amplified from IMAGE EST 3875593 using Expand HiFi Polymerase (Roche), then cloned into pCR2.1 (Invitrogen) and sequenced to completion. The resulting fragment was digested with BamH1 and Not1 and cloned into the same sites in pGEX6P-1 to create pGEX6P-1 IL-1β[117–268]. Site-directed mutagenesis of Tpl2 was carried out using the Stratagene Quikchange® protocol. The sequences of all clones were verified by the DNA Sequencing Service in the MRC Protein Phosphorylation Unit (http://www.dnaseq.co.uk).

Cell culture, transfection and lysis

HEK-293 cells stably expressing the IL-1R, termed IL-1R cells, and IRAK1-null IL-1R cells (provided by Dr Xiaoxia Li and Dr George Stark, The Cleveland Clinic Foundation, Cleveland, OH, U.S.A.) [22], were cultured in DMEM (Dulbecco's modified Eagle's medium) with 10% (v/v) FCS (foetal calf serum). Cells were transfected at 40–50% confluency using polyethyleneimine and 0.5 μg (Tpl2T), 1 μg (Tpl2L and Tpl2S) or 2 μg (catalytically inactive mutants of Tpl2L and Tpl2L[T290A]) of DNA. The FLAG–Tpl2 was expressed at a lower level than the endogenous Tpl2 in all of the experiments reported in the present paper. After 24 h, the medium was replaced with DMEM without serum and then left for 16 h before stimulation with 2.5 ng/ml IL-1β. All cells were maintained at 37°C in a 95% air and 5% CO2 atmosphere and lysed in ice-cold lysis buffer [50 mM Tris/HCl (pH 7.5), 1 mM EDTA, 1 mM EGTA, 1 mM sodium orthovanadate, 10 mM sodium β-glycerolphosphate, 50 mM sodium fluoride, 5 mM sodium pyrophosphate, 0.27 M sucrose, 1% (v/v) Triton X-100, 0.1% (v/v) 2-mercaptoethanol and Complete™ proteinase inhibitor cocktail (Roche: one tablet per 50 ml of buffer)]. Lysates were centrifuged at 13000 g for 15 min at 4°C and the supernatants used immediately or snap-frozen in liquid nitrogen and stored in aliquots at −20°C until use. In experiments where protein kinase inhibitors were used, aliquots of these compounds dissolved at 10 mM in DMSO were added to the cell culture medium and an equivalent volume of DMSO was added to control incubations.

Immunoprecipitation of tagged proteins and immunoblotting

FLAG–Tpl2 and HA–ABIN2 expressed in IL-1R cells were immunoprecipitated with anti-FLAG–agarose and anti-HA–agarose respectively. To immunoprecipitate FLAG–Tpl2 or HA–ABIN2, 5 μl of packed anti-FLAG–agarose or anti-HA–agarose beads were added to 0.5–1.0 mg of cell lysate protein and incubated for 60 min at 4°C on a shaking platform. After centrifugation for 15 s at 13000 g, the supernatant was removed and the beads washed twice with 1 ml of lysis buffer. This was followed by two washes with 1 ml of Buffer A [10 mM Tris/HCl (pH 7.5), 2 mM EDTA and 0.2% (v/v) Nonidet P40] plus 150 mM NaCl, two washes with 1 ml of Buffer A plus 500 mM NaCl and three washes with 1 ml of 10 mM Tris/HCl (pH 7.5). The immunoprecipitates were then denatured in SDS to release proteins from the antibody, and subjected to SDS/PAGE. After transfer on to nitrocellulose, the membranes were immunoblotted using the ECL® detection system (GE Healthcare).

Immunoprecipitation and assay of Tpl2

The endogenous Tpl2 in cell extracts was immunoprecipitated by coupling 1 μg of Tpl2 antibody non-covalently to 5 μl of Protein G–Sepharose beads. Cell extract (1–2 mg of protein) was added and, after incubation for 2 h at 4°C on a shaking platform, the beads were collected and washed as described above. The immunoprecipitated endogenous Tpl2 or immunoprecipitated FLAG–Tpl2 from transfection experiments were assayed and the activity quantified as described previously for c-Raf [23] using a thermomixer to keep the beads agitated. In this two-step assay, Tpl2 is assayed by its ability to activate MKK1, which is then assayed by the activation of ERK2. The active ERK2 is then assayed by the phosphorylation of MBP (myelin basic protein). In some experiments, the Tpl2 immunoprecipitates were incubated for 30 min at 30°C with phage λ phosphatase in 50 mM Hepes (pH 7.5), 100 mM NaCl, 2 mM DTT (dithiothreitol), 0.01% (w/v) Brij-35 and 1 mM MnCl2 in a total volume of 50 μl. The reaction was agitated continuously and stopped by making the solution 1 mM EGTA, 10 mM sodium fluoride, 25 mM β-glycerolphosphate and 0.1 mM sodium orthovanadate. The immunoprecipitates were then washed five times with 1 ml of 10 mM Tris/HCl (pH 7.5) and assayed for Tpl2 activity.

RESULTS

IRAK1 is required for the IL-1-stimulated phosphorylation of p105 and the activation of the endogenous Tpl2 complex in IL-1R cells

The activation of the transcription factor NF-κB and MAPKs by IL-1 is known to require the IRAK4-catalysed activation of IRAK1, which leads to the activation of IKKβ and hence to the phosphorylation of its substrates IκBα and p105 (reviewed in [24]). The phosphorylation of IκBα triggers its proteasomal destruction thereby activating NF-κB, whereas the phosphorylation of p105 leads to the activation of Tpl2.

Consistent with the key role played by IRAK1 in the activation of IKKβ, the IL-1-stimulated phosphorylation of IKKα/β and degradation of IκBα was greatly reduced and the phosphorylation of p105 abolished in IRAK1-null HEK-293 cells that stably express the IL-1R (Figure 1A). The IL-1-stimulated activation of the endogenous Tpl2 complex (Figure 1B) and the activation of ERK1/ERK2 (Figure 1A) were also greatly reduced in the IRAK1-null cells, presumably as a result of the lack of phosphorylation of p105. The slight residual activation may be explained by the presence of low levels of the IRAK2 isoform in these cells (X. Li, personal communication). Consistent with the known role of IKKβ, the IKKβ inhibitor PS1145 suppressed the IL-1-stimulated activation of the endogenous Tpl2 complex (Figure 1B) and greatly reduced the activation of ERK1/ERK2 in the control IL-1R cells that express IRAK1 (Figure 1C). As expected, PS1145 also prevented the degradation of IκBα and the phosphorylation of p105 (Figure 1C), and did not affect the IL-1-stimulated phosphorylation (activation) of p38α MAPK and JNK (Figure 1C).

Components of IL-1-stimulated signalling pathways that are dependent on the presence of IRAK1 and the activity of IKKβ in IL-1R cells

Figure 1
Components of IL-1-stimulated signalling pathways that are dependent on the presence of IRAK1 and the activity of IKKβ in IL-1R cells

(A) Wild-type IL-1R cells (IRAK1+/+) and IRAK1-null IL-1R cells (IRAK1−/−) were serum-starved for 16 h in DMEM, before stimulation with 2.5 ng/ml IL-1 for the times indicated. Following cell lysis and SDS/PAGE, immunoblotting was carried out with antibodies that recognize IRAK1, IKKα/β phosphorylated at Ser180 (IKKα) and Ser181 (IKKβ), IκBα, p105 phosphorylated at Ser933 (p-p105), the phosphorylated Thr-Glu-Tyr motif of ERK1 and ERK2 (p-ERK1 and p-ERK2) and all forms of ERK1/ERK2. The IL-1-induced ‘disappearance’ of IRAK1 may result from its conversion into a variety of polyubiquitylated species [29]. (B) The experiment was carried out as in (A) except that, where indicated, the serum-starved cells were incubated for 60 min without or with 15 μM PS1145, prior to stimulation for 15 min with 2.5 ng/ml IL-1. Following cell lysis, the endogenous Tpl2 was immunoprecipitated from 1 mg of cell extract protein with 1 μg of anti-Tpl2. Protein G–Sepharose (15 μl of packed beads) was added and, after mixing for 1 h, the beads were collected, washed and assayed for Tpl2 activity. Values are means±S.D. for three different immunoprecipitations from one experiment and are expressed as units of activity per mg of cell lysate protein. Similar results were obtained in two other independent experiments. (C) Wild-type IL-1R cells were serum-starved for 16 h in DMEM, then incubated for 1 h in the absence (−) or presence (+) of 15 μM PS1145. After stimulation for 15 min with 2.5 ng/ml IL-1, 25 μg of cell extract protein was denatured in SDS, subjected to SDS/PAGE, transferred on to nitrocellulose and immunoblotted with the antibodies described in (A) plus further antibodies that recognize the phosphorylated Thr-Pro-Tyr motif of JNK1/JNK2 (p-JNK1 and p-JNK2), and the phosphorylated Thr-Gly-Tyr motif of p38α MAPK (p-p38α MAPK). As a loading control, the same lysate was also immunoblotted with an antibody that recognizes the phosphorylated and unphosphorylated forms of p38α MAPK equally well.

Figure 1
Components of IL-1-stimulated signalling pathways that are dependent on the presence of IRAK1 and the activity of IKKβ in IL-1R cells

(A) Wild-type IL-1R cells (IRAK1+/+) and IRAK1-null IL-1R cells (IRAK1−/−) were serum-starved for 16 h in DMEM, before stimulation with 2.5 ng/ml IL-1 for the times indicated. Following cell lysis and SDS/PAGE, immunoblotting was carried out with antibodies that recognize IRAK1, IKKα/β phosphorylated at Ser180 (IKKα) and Ser181 (IKKβ), IκBα, p105 phosphorylated at Ser933 (p-p105), the phosphorylated Thr-Glu-Tyr motif of ERK1 and ERK2 (p-ERK1 and p-ERK2) and all forms of ERK1/ERK2. The IL-1-induced ‘disappearance’ of IRAK1 may result from its conversion into a variety of polyubiquitylated species [29]. (B) The experiment was carried out as in (A) except that, where indicated, the serum-starved cells were incubated for 60 min without or with 15 μM PS1145, prior to stimulation for 15 min with 2.5 ng/ml IL-1. Following cell lysis, the endogenous Tpl2 was immunoprecipitated from 1 mg of cell extract protein with 1 μg of anti-Tpl2. Protein G–Sepharose (15 μl of packed beads) was added and, after mixing for 1 h, the beads were collected, washed and assayed for Tpl2 activity. Values are means±S.D. for three different immunoprecipitations from one experiment and are expressed as units of activity per mg of cell lysate protein. Similar results were obtained in two other independent experiments. (C) Wild-type IL-1R cells were serum-starved for 16 h in DMEM, then incubated for 1 h in the absence (−) or presence (+) of 15 μM PS1145. After stimulation for 15 min with 2.5 ng/ml IL-1, 25 μg of cell extract protein was denatured in SDS, subjected to SDS/PAGE, transferred on to nitrocellulose and immunoblotted with the antibodies described in (A) plus further antibodies that recognize the phosphorylated Thr-Pro-Tyr motif of JNK1/JNK2 (p-JNK1 and p-JNK2), and the phosphorylated Thr-Gly-Tyr motif of p38α MAPK (p-p38α MAPK). As a loading control, the same lysate was also immunoblotted with an antibody that recognizes the phosphorylated and unphosphorylated forms of p38α MAPK equally well.

IL-1 activates the transfected Tpl2 catalytic subunit in IRAK1-null cells

We have reported previously that IL-1 can induce the activation of a truncated form of the Tpl2 catalytic subunit (Tpl2T) in transfected IL-1R cells [18], and similar results were obtained in the present study with the full length, long form of Tpl2 (Tpl2L) (Figure 2A). Surprisingly, IL-1 also induced a similar activation of transfected Tpl2L in the IRAK1-null IL-1R cells (Figure 2A), where IKKβ is not activated (Figures 1A, 2A and 2B), demonstrating that IL-1 activates Tpl2L in IRAK1-null IL-1R cells by a mechanism that is independent of IRAK1, IKKβ and the phosphorylation of p105.

The IL-1-stimulated activation of transfected Tpl2L is independent of the presence of IRAK1 and the activity of IKKβ

Figure 2
The IL-1-stimulated activation of transfected Tpl2L is independent of the presence of IRAK1 and the activity of IKKβ

(A) DNA encoding FLAG–Tpl2L was transfected into wild-type IL-1R cells (IRAK+/+) or IRAK1-null IL-1R cells (IRAK−/−). After 24 h, the cells were incubated for a further 16 h in DMEM without serum and then for 1 h with (+) or without (−) 15 μM PS1145 before stimulation for 15 min with (+) or without (−) 2.5 ng/ml IL-1. Following cell lysis, the transfected Tpl2 was immunoprecipitated from 0.5 mg of cell extract protein using anti-FLAG–agarose beads. The beads were collected by centrifugation and then washed and assayed for Tpl2 activity. Values are means±S.D. for three different immunoprecipitations from one experiment and are expressed as units of activity per mg of cell lysate protein. Similar results were obtained in two other independent experiments. (B) The experiment was carried out as in (A) using only the IRAK1-null cells. Cell lysate protein (25 μg) was subjected to immunoblotting with the antibodies described in the legend to Figure 1(A) and with anti-FLAG (FLAG) to detect the expression of the transfected FLAG–Tpl2L.

Figure 2
The IL-1-stimulated activation of transfected Tpl2L is independent of the presence of IRAK1 and the activity of IKKβ

(A) DNA encoding FLAG–Tpl2L was transfected into wild-type IL-1R cells (IRAK+/+) or IRAK1-null IL-1R cells (IRAK−/−). After 24 h, the cells were incubated for a further 16 h in DMEM without serum and then for 1 h with (+) or without (−) 15 μM PS1145 before stimulation for 15 min with (+) or without (−) 2.5 ng/ml IL-1. Following cell lysis, the transfected Tpl2 was immunoprecipitated from 0.5 mg of cell extract protein using anti-FLAG–agarose beads. The beads were collected by centrifugation and then washed and assayed for Tpl2 activity. Values are means±S.D. for three different immunoprecipitations from one experiment and are expressed as units of activity per mg of cell lysate protein. Similar results were obtained in two other independent experiments. (B) The experiment was carried out as in (A) using only the IRAK1-null cells. Cell lysate protein (25 μg) was subjected to immunoblotting with the antibodies described in the legend to Figure 1(A) and with anti-FLAG (FLAG) to detect the expression of the transfected FLAG–Tpl2L.

IL-1 induced a similar degree of activation of the transfected short form of Tpl2 (Tpl2S) and Tpl2T in IRAK1-null IL-1R cells, but the kinetics of activation were quite different (Supplementary Figure S1 at http://www.BiochemJ.org/bj/424/bj4240109add.htm). Tpl2L Tpl2S and Tpl2T were activated maximally at 15, 45 and 30 min after stimulation with IL-1 respectively, with activity declining thereafter, as judged by the activation of ERK1/ERK2 (Supplementary Figure S1) or direct measurement of Tpl2 activity (results not shown).

The Src inhibitor PP2 prevents the IL-1-induced activation and phosphorylation of the transfected Tpl2 catalytic subunit by a Src-independent pathway

The IL-1-stimulated activation of ERK1/ERK2 in HeLa cells was reported to be suppressed by the inclusion of PP1 or PP2 in the culture medium [8]. These compounds were originally developed as small molecule inhibitors of the Src family of protein tyrosine kinases [25]. They do not affect the activity of Tpl2T or its downstream target MKK1 in vitro, even at 0.1 mM (L. Plater and P. Cohen, unpublished work). In the present study, we found that PP2 (25 μM) suppressed the IL-1-stimulated activation of the endogenous Tpl2 complex (Figure 3A), as well as the activation of ERK1/ERK2 (Figure 3B) in the IRAK1-expressing IL-IR cells. Importantly, PP2 did not prevent the IL-1-induced degradation of IκBα or the phosphorylation of p105 (Figure 3B), showing that PP2 had not exerted its effect by inhibiting the activation or activity of IKKβ. PP2 also did not affect the IL-1-stimulated phosphorylation (activation) of p38α MAPK and JNK (Figure 3B).

PP2 inhibits the IL-1-stimulated activation of endogenous Tpl2 in IRAK1+/+ IL-1R cells

Figure 3
PP2 inhibits the IL-1-stimulated activation of endogenous Tpl2 in IRAK1+/+ IL-1R cells

(A) IL-1R cells were serum-starved for 16 h, incubated with the indicated concentrations of PP2, stimulated for 15 min with (+) or without (−) IL-1 and the endogenous Tpl2 was immunoprecipitated and assayed as described in the legend to Figure 1(B). (B) Cell lysate protein (25 μg) from (A) was subjected to SDS/PAGE, transferred on to a nitrocellulose membrane and immunoblotted with the antibodies described in the legend to Figures 1(A) and 1(C), as well as with an antibody that recognizes p130CAS phosphorylated at Tyr410 (p-p130CAS). (C) IRAK1-null IL-1R cells were serum-starved for 16 h in DMEM, then incubated for 1 h with the indicated concentrations of PP2 prior to cell lysis (no stimulation with IL-1). Cell extract protein (25 μg) was immunoblotted with antibodies that recognize the phosphorylated forms of p130CAS (p-p130CAS) and all forms of ERK1/ERK2.

Figure 3
PP2 inhibits the IL-1-stimulated activation of endogenous Tpl2 in IRAK1+/+ IL-1R cells

(A) IL-1R cells were serum-starved for 16 h, incubated with the indicated concentrations of PP2, stimulated for 15 min with (+) or without (−) IL-1 and the endogenous Tpl2 was immunoprecipitated and assayed as described in the legend to Figure 1(B). (B) Cell lysate protein (25 μg) from (A) was subjected to SDS/PAGE, transferred on to a nitrocellulose membrane and immunoblotted with the antibodies described in the legend to Figures 1(A) and 1(C), as well as with an antibody that recognizes p130CAS phosphorylated at Tyr410 (p-p130CAS). (C) IRAK1-null IL-1R cells were serum-starved for 16 h in DMEM, then incubated for 1 h with the indicated concentrations of PP2 prior to cell lysis (no stimulation with IL-1). Cell extract protein (25 μg) was immunoblotted with antibodies that recognize the phosphorylated forms of p130CAS (p-p130CAS) and all forms of ERK1/ERK2.

To investigate whether PP2 inhibited the activation of Tpl2 by a Src-dependent mechanism we studied the phosphorylation of p130CAS, which is phosphorylated at Tyr410 by Src family members [26]. The phosphorylation of p130CAS was completely suppressed at 1.0 μM PP2 (Figure 3B), a concentration that had no effect on the IL-1-stimulated activation of the endogenous Tpl2 complex (Figure 3A) or the activation of ERK1/ERK2 (Figure 3B). Indeed, further studies showed that PP2 prevented the phosphorylation of p130CAS with an IC50 below 0.2 μM (Figure 3C). Similar results were obtained in IRAK1-null IL-1R cells, PP2 suppressing the IL-1-stimulated activation of Tpl2L, Tpl2S and Tpl2T (Figure 4A) and consequent activation of ERK1/ERK2 (Figure 4B) at 25 μM, but not 1 μM, PP2. These results suggested that PP2 exerts its effect on the IL-1-stimulated activation of Tpl2 by a Src-independent pathway. Similar results were obtained with the structurally unrelated Src inhibitor SU6656, which blocked the activation of p130CAS at concentrations that did not affect the IL-1-stimulated activation of transfected Tpl2 in IRAK1-null cells (Supplementary Figures S2A and S2B at http://www.BiochemJ.org/bj/424/bj4240109add.htm). These results confirmed that Src does not mediate the IL-1-stimulated activation of the Tpl2 catalytic subunit.

Effect of Src family kinase inhibitors on the IL-1-stimulated activation of transfected Tpl2 in IRAK1-null IL-1R cells

Figure 4
Effect of Src family kinase inhibitors on the IL-1-stimulated activation of transfected Tpl2 in IRAK1-null IL-1R cells

(A) IRAK1-null IL-1R cells were transfected with DNA encoding FLAG–Tpl2L, FLAG–Tpl2S or FLAG–Tpl2T. After 24 h, the cells were serum-starved in DMEM for a further 16 h, then incubated for 60 min with the indicated concentrations of PP2, and then for a further 15 min with (+) or without (−) 2.5 ng/ml IL-1. After cell lysis, FLAG–Tpl2 was immunoprecipitated from 0.5 mg of cell extract protein with anti-FLAG–agarose beads and assayed for Tpl2 activity as described in the legend to Figure 2(A). Values are means±S.D. for three different immunoprecipitations from one experiment and are expressed as units of activity per mg of cell lysate protein. Similar results were obtained in another independent experiment. (B) Cell lysate protein (25 μg) from (A) was subjected to SDS/PAGE, transferred on to a nitrocellulose membrane and immunoblotted with antibodies that recognize the phosphorylated forms of ERK1/ERK2 and with anti-FLAG to detect the transfected Tpl2.

Figure 4
Effect of Src family kinase inhibitors on the IL-1-stimulated activation of transfected Tpl2 in IRAK1-null IL-1R cells

(A) IRAK1-null IL-1R cells were transfected with DNA encoding FLAG–Tpl2L, FLAG–Tpl2S or FLAG–Tpl2T. After 24 h, the cells were serum-starved in DMEM for a further 16 h, then incubated for 60 min with the indicated concentrations of PP2, and then for a further 15 min with (+) or without (−) 2.5 ng/ml IL-1. After cell lysis, FLAG–Tpl2 was immunoprecipitated from 0.5 mg of cell extract protein with anti-FLAG–agarose beads and assayed for Tpl2 activity as described in the legend to Figure 2(A). Values are means±S.D. for three different immunoprecipitations from one experiment and are expressed as units of activity per mg of cell lysate protein. Similar results were obtained in another independent experiment. (B) Cell lysate protein (25 μg) from (A) was subjected to SDS/PAGE, transferred on to a nitrocellulose membrane and immunoblotted with antibodies that recognize the phosphorylated forms of ERK1/ERK2 and with anti-FLAG to detect the transfected Tpl2.

Phosphorylation of the Tpl2 catalytic subunit during activation by IL-1

We have reported previously that the IL-1-stimulated activation of transfected Tpl2T is accompanied by its phosphorylation at Ser62 and Thr290 in IRAK1-expressing IL-1R cells [18]. In the present study, we found that IL-1 also stimulated the phosphorylation of transfected Tpl2T at these two sites in IRAK1-null IL-1R cells, and that IL-1 also induced the phosphorylation of Tpl2L and Tpl2S at Ser400, as well as at Ser62 and Thr290 (Figure 5). Importantly, PP2 prevented the IL-1-stimulated phosphorylation of Tpl2 at Thr290 and Ser400, and reduced the phosphorylation of Ser62 to the basal level observed in the absence of IL-1 stimulation (Figure 5).

PP2 prevents the IL-1-stimulated phosphorylation of transfected Tpl2 in IRAK1-null IL-1R cells

Figure 5
PP2 prevents the IL-1-stimulated phosphorylation of transfected Tpl2 in IRAK1-null IL-1R cells

The experiment was carried out using IRAK1-null IL-1R cells as described in the legend to Figure 4(A) after transfection with DNA encoding the three forms of Tpl2. Following cell lysis, FLAG–Tpl2 was immunoprecipitated from 0.5 mg of cell extract protein with anti-FLAG beads. After release from the anti-FLAG beads by denaturation in SDS, the samples were subjected to SDS/PAGE, transferred on to a PVDF membrane and immunoblotted with antibodies that recognize the phosphorylated forms of ERK1/ERK2 and Tpl2 phosphorylated at Ser62, Thr290 and Ser400 respectively. The same membrane was also immunoblotted with anti-FLAG as a loading control. The positions at which FLAG–Tpl2L, FLAG–Tpl2S and FLAG–Tpl2T migrate are denoted by L, S and T. IP, immunoprecipitation.

Figure 5
PP2 prevents the IL-1-stimulated phosphorylation of transfected Tpl2 in IRAK1-null IL-1R cells

The experiment was carried out using IRAK1-null IL-1R cells as described in the legend to Figure 4(A) after transfection with DNA encoding the three forms of Tpl2. Following cell lysis, FLAG–Tpl2 was immunoprecipitated from 0.5 mg of cell extract protein with anti-FLAG beads. After release from the anti-FLAG beads by denaturation in SDS, the samples were subjected to SDS/PAGE, transferred on to a PVDF membrane and immunoblotted with antibodies that recognize the phosphorylated forms of ERK1/ERK2 and Tpl2 phosphorylated at Ser62, Thr290 and Ser400 respectively. The same membrane was also immunoblotted with anti-FLAG as a loading control. The positions at which FLAG–Tpl2L, FLAG–Tpl2S and FLAG–Tpl2T migrate are denoted by L, S and T. IP, immunoprecipitation.

Evidence that the phosphorylation of Ser62, Thr290 and Ser400 are autophosphorylation events

To investigate whether the phosphorylation of Tpl2L and Tpl2T was catalysed by a distinct protein kinase(s) or by Tpl2 itself, we transfected IRAK1-null cells with the catalytically inactive mutants Tpl2[D270A] and Tpl2[K167R]. As expected, these mutants displayed no activity, even after stimulation with IL-1 (Supplementary Figure S3 at http://www.BiochemJ.org/bj/424/bj4240109add.htm), and were unable to restore IL-1-stimulated activation of ERK1/ERK2 to IRAK1-null IL-1R cells (Figure 6, top panel). Moreover, in contrast with wild-type Tpl2, IL-1 was unable to enhance the phosphorylation of Ser62 or induce any phosphorylation of Thr290 or Ser400 in either of the catalytically inactive mutants (Figure 6, bottom three panels). This suggested that the phosphorylation of all three residues was catalysed by Tpl2 itself, after it had been activated. In contrast, the basal phosphorylation of Ser62 in unstimulated cells was unaffected, demonstrating that it is catalysed by a distinct protein kinase (Figure 6, middle panel).

Catalytically inactive mutants of transfected Tpl2 are not phosphorylated at Thr290 and Ser400 in IRAK1-null IL-1R cells

Figure 6
Catalytically inactive mutants of transfected Tpl2 are not phosphorylated at Thr290 and Ser400 in IRAK1-null IL-1R cells

The indicated DNA constructs were transfected into IRAK1-null IL-1R cells and after 24 h the cells were serum-starved in DMEM for a further 16 h. After stimulation for 15 min with (+) or without (−) 2.5 ng/ml IL-1, the cells were lysed and subjected to immunoblotting as described in the legend to Figure 5. The positions at which FLAG–Tpl2L and FLAG–Tpl2T migrate are denoted by L and T respectively. IP, immunoprecipitation; WT, wild-type.

Figure 6
Catalytically inactive mutants of transfected Tpl2 are not phosphorylated at Thr290 and Ser400 in IRAK1-null IL-1R cells

The indicated DNA constructs were transfected into IRAK1-null IL-1R cells and after 24 h the cells were serum-starved in DMEM for a further 16 h. After stimulation for 15 min with (+) or without (−) 2.5 ng/ml IL-1, the cells were lysed and subjected to immunoblotting as described in the legend to Figure 5. The positions at which FLAG–Tpl2L and FLAG–Tpl2T migrate are denoted by L and T respectively. IP, immunoprecipitation; WT, wild-type.

To investigate further whether Tpl2 was really capable of autophosphorylation at Ser62, Thr290 and Ser400, we immunoprecipitated transfected Tpl2L from the lysates of control and IL-1-stimulated IRAK1-null IL-1R cells. These preparations, which were phosphorylated at Ser62 or at Ser62, Thr290 and Ser400 respectively (Figure 7A, lanes 1 and 2) were then incubated with the protein phosphatase encoded by phage λ, which led to the dephosphorylation of all three sites (Figure 7A, lanes 5 and 6). Incubation of the phosphatase-treated Tpl2L immunoprecipitates with Mg-ATP led to the rephosphorylation of Ser62 (Tpl2L from control cells) or Ser62, Thr290 and Ser400 (Tpl2L from IL-1-stimulated cells) (Figure 7A, lanes 7 and 8). The rephosphorylation of Ser62, Thr290 and Ser400 by Tpl2L immunoprecipitates from IL-1stimulated cells was prevented by C-1 (Figure 7A, lane 10), a cell-permeable inhibitor of Tpl2 [9], indicating that activated Tpl2 is indeed capable of autophosphorylation at Thr290 and Ser400. However, the rephosphorylation of phosphatase-treated Tpl2L was unaffected by PP2 (Figure 7A, lane 12), consistent with the failure of this compound to inhibit Tpl2 activity in vitro, and indicating that it prevents the IL-1-stimulated activation of Tpl2 by targeting a more upstream component of the signalling pathway.

Dephosphorylation and rephosphorylation of Tpl2 at Ser62, Thr290 and Ser400

Figure 7
Dephosphorylation and rephosphorylation of Tpl2 at Ser62, Thr290 and Ser400

(A) FLAG–Tpl2L was transfected into IRAK1-null IL-1R cells and stimulated for 15 min with (+) or without (−) 2.5 ng/ml IL-1. After cell lysis, FLAG–Tpl2 was immunoprecipitated from 1 mg of cell extract protein with anti-FLAG–agarose and the washed beads incubated for 30 min at 30°C with (+) or without (−) 4 units of phage λ phosphatase (λPPase). After washing to remove the phosphatase, the immunoprecipitates were incubated for 30 min at 30°C without (−) or with (+) C-1 (10 μM) or PP2 (25 μM), and then for 30 min at 30°C with 10 mM MgCl2 and 0.1 mM ATP in the standard Tpl2 assay buffer. The reactions were stopped by denaturation in SDS, followed by SDS/PAGE and immunoblotting with antibodies that recognize Tpl2 phosphorylated at Ser62, Thr290 and Ser400 and with anti-FLAG to detect Tpl2. (B) Same as (A), except that the protein kinase inhibitor staurosporine was used at 0.1 μM instead of C-1 or PP2.

Figure 7
Dephosphorylation and rephosphorylation of Tpl2 at Ser62, Thr290 and Ser400

(A) FLAG–Tpl2L was transfected into IRAK1-null IL-1R cells and stimulated for 15 min with (+) or without (−) 2.5 ng/ml IL-1. After cell lysis, FLAG–Tpl2 was immunoprecipitated from 1 mg of cell extract protein with anti-FLAG–agarose and the washed beads incubated for 30 min at 30°C with (+) or without (−) 4 units of phage λ phosphatase (λPPase). After washing to remove the phosphatase, the immunoprecipitates were incubated for 30 min at 30°C without (−) or with (+) C-1 (10 μM) or PP2 (25 μM), and then for 30 min at 30°C with 10 mM MgCl2 and 0.1 mM ATP in the standard Tpl2 assay buffer. The reactions were stopped by denaturation in SDS, followed by SDS/PAGE and immunoblotting with antibodies that recognize Tpl2 phosphorylated at Ser62, Thr290 and Ser400 and with anti-FLAG to detect Tpl2. (B) Same as (A), except that the protein kinase inhibitor staurosporine was used at 0.1 μM instead of C-1 or PP2.

The finding that the inactive Tpl2L became rephosphorylated only at Ser62 upon incubation with Mg-ATP (Figure 7A, lane 7) suggested that it was contaminated with a protein kinase distinct from Tpl2L that phosphorylates Ser62in vitro. To investigate this possibility we used staurosporine, a potent inhibitor of many protein kinases, which we have found does not inhibit Tpl2. Staurosporine prevented the phosphorylation of Ser62 by the inactive Tpl2L immunoprecipitates from control cells (Figure 7B, compare lanes 5 and 6), but had no effect on the phosphorylation of Ser62, Thr290 and Ser400 by the active Tpl2 immunoprecipitates from IL-1-stimulated cells (Figure 7B, compare lanes 7 and 8). These experiments established that Tpl2 catalyses the autophosphorylation of Ser62, Thr290 and Ser400 and confirmed that Ser62 phosphorylation by inactive Tpl2 immunoprecipitates is catalysed by a distinct protein kinase.

The phosphorylation of Ser62 by inactive Tpl2 immunoprecipitates was prevented by C-1 (Figure 7A, lane 9), showing that the binding of C-1 to Tpl2 prevents the phosphorylatiion of Ser62 by the contaminating protein kinase.

Site-directed mutagenesis of the phosphorylation sites on Tpl2

The incubation of Tpl2 immunoprecipitates from IL-1-stimulated cells with phage λ phosphatase had little effect on the ability of Tpl2 to activate MKK1 (Supplementary Figure S4 at http://www.BiochemJ.org/bj/424/bj4240109add.htm). However, as the phosphatase-treated Tpl2 became rephosphorylated at Ser62, Thr290 and Ser400 within minutes of incubation with Mg-ATP (results not shown), this presumably occurs during the standard assay of Tpl2 making it impossible to assess whether the phosphorylation of these sites is required for the activation of MKK1. To address this issue we therefore mutated these sites to alanine and studied whether the mutant enzymes could be activated by IL-1 in transfected IRAK1-null cells.

The mutation of Ser400 to alanine only decreased the IL-1-stimulated activation of Tpl2L slightly, whereas the mutation of Ser62 to alanine reduced activation by 40–50% (Figure 8A). In contrast, the mutation of Thr290 to alanine in Tpl2L virtually abolished the activation of MKK1 by Tpl2 in vitro (Figure 8A), as well as the IL-1-stimulated activation of ERK1/ERK2 in transfected cells (Figure 8B). Interestingly, the mutation of both Ser62 and Ser400 to alanine prevented the IL-1-stimulated phosphorylation of Thr290 (Figure 8B) and the activation of Tpl2 (Figure 8A). Taken together, these results suggest that the autophosphorylation of Tpl2 at Thr290 may be needed for Tpl2 to activate MKK1.

Effect of mutating phosphorylation sites on the IL-1-stimulated activation of Tpl2L in transfected IRAK1-null IL-1R cells

Figure 8
Effect of mutating phosphorylation sites on the IL-1-stimulated activation of Tpl2L in transfected IRAK1-null IL-1R cells

(A) IRAK1-null IL-1R cells were transfected with the indicated DNA constructs and, after 24 h, were serum-starved in DMEM for a further 16 h. The cells were then stimulated for 15 min with (+) or without (−) 2.5 ng/ml IL-1. Following cell lysis, FLAG–Tpl2L was immunoprecipitated from 1.0 mg of cell extract with anti-FLAG–agarose beads and assayed for activity as described in the legend to Figure 2(A). (B) The immunoprecipitates from (A) were denatured in SDS, subjected to SDS/PAGE and immunoblotted with the antibodies described in the legend to Figure 5. IP, immunoprecipitation; WT, wild-type.

Figure 8
Effect of mutating phosphorylation sites on the IL-1-stimulated activation of Tpl2L in transfected IRAK1-null IL-1R cells

(A) IRAK1-null IL-1R cells were transfected with the indicated DNA constructs and, after 24 h, were serum-starved in DMEM for a further 16 h. The cells were then stimulated for 15 min with (+) or without (−) 2.5 ng/ml IL-1. Following cell lysis, FLAG–Tpl2L was immunoprecipitated from 1.0 mg of cell extract with anti-FLAG–agarose beads and assayed for activity as described in the legend to Figure 2(A). (B) The immunoprecipitates from (A) were denatured in SDS, subjected to SDS/PAGE and immunoblotted with the antibodies described in the legend to Figure 5. IP, immunoprecipitation; WT, wild-type.

Although the mutation of Thr290 to alanine prevented Tpl2 from activating MKK1, it did not prevent the autophosphorylation of Tpl2 at Ser62 or Ser400 (Figure 8B). Taken together, these experiments demonstrate that the phosphorylation of Thr290 is not required for the IL-1-induced conformational change that allows Tpl2 to autophosphorylate, although it may be needed for Tpl2 to phosphorylate and activate MKK1.

We also carried out mutagenesis studies on Tpl2T (Supplementary Figures S5A and S5B at http://www.BiochemJ.org/bj/424/bj4240109add.htm). This species was activated by IL-1, in a similar manner to Tpl2L (Supplementary Figure S1B), demonstrating that the phosphorylation of Ser400 or any other residue in the C-terminal tail between amino acid residues 398 and 467 is not required for the IL-1-induced conformation change that activates the Tpl2 catalytic subunit. As reported previously [18], the mutation of Ser62 to alanine only reduced the IL-1-stimulated activation of Tpl2T by 40% (Supplementary Figure S5A), demonstrating that IL-1 can still activate a form of the Tpl2 catalytic subunit in which neither Ser62 nor Ser400 are phosphorylated. Similar to Tpl2L, the mutation of Thr290 to alanine prevented the IL-1-stimulated activation of Tpl2T, but in contrast with Tpl2L, it also prevented the phosphorylation of Ser62 (Supplementary Figure S5B).

The mutagenesis experiments presented in Figure 8(B) also established the specificity of the phospho-specific antibodies used in these experiments. The mutation of Ser62 to alanine prevented recognition of this site by the anti-phospho-Ser62 antibody without affecting the IL-1-stimulated phosphorylation of Thr290 or Ser400. Similarly, mutagenesis of Thr290 to alanine prevented recognition by the anti-phospho-Thr290 antibody without affecting the IL-1-stimulated phosphorylation of Ser62 or Ser400, whereas mutagenesis of Ser400 to alanine prevented recognition by the anti-phospho-Ser400 antibody without affecting the phosphorylation of Ser62 or Thr290 (Figure 9B).

The IL-1-stimulated dissociation of ABIN2 from Tpl2 in IRAK1-null IL-1R cells does not require Tpl2 activity

Figure 9
The IL-1-stimulated dissociation of ABIN2 from Tpl2 in IRAK1-null IL-1R cells does not require Tpl2 activity

(A) DNA encoding wild-type (WT) FLAG–Tpl2L or the indicated Tpl2 mutants were co-transfected into IRAK1-null IL-1R cells with HA–ABIN2. After 24 h, the cells were serum-starved in DMEM for 16 h, incubated for 60 min without (−) or with (+) 10 μM C-1, then stimulated for 15 min with (+) or without (−) 2.5 ng/ml IL-1. Following cell lysis, 25 μg of cell extract protein was immunoblotted to detect the phosphorylated forms of ERK1/ERK2 (p-ERK1 and p-ERK2). FLAG–Tpl2 or HA–ABIN2 were immunoprecipitated (IP) from a further 1 mg of cell extract protein using anti-FLAG–agarose or anti-HA–Protein G–Sepharose respectively. The beads were washed and bound proteins released from the antibodies with 0.1% SDS, subjected to SDS/PAGE and immunoblotted with anti-FLAG to detect Tpl2 and anti-HA to detect ABIN2. The immunoprecipitated Tpl2 was also immunoblotted with the antibodies described in the legend to Figure 5 to detect phosphorylation at Ser62, Thr290 and Ser400. (B) The experiment was carried out as described in (A) except that only wild-type Tpl2L was used and PP2 replaced C-1.

Figure 9
The IL-1-stimulated dissociation of ABIN2 from Tpl2 in IRAK1-null IL-1R cells does not require Tpl2 activity

(A) DNA encoding wild-type (WT) FLAG–Tpl2L or the indicated Tpl2 mutants were co-transfected into IRAK1-null IL-1R cells with HA–ABIN2. After 24 h, the cells were serum-starved in DMEM for 16 h, incubated for 60 min without (−) or with (+) 10 μM C-1, then stimulated for 15 min with (+) or without (−) 2.5 ng/ml IL-1. Following cell lysis, 25 μg of cell extract protein was immunoblotted to detect the phosphorylated forms of ERK1/ERK2 (p-ERK1 and p-ERK2). FLAG–Tpl2 or HA–ABIN2 were immunoprecipitated (IP) from a further 1 mg of cell extract protein using anti-FLAG–agarose or anti-HA–Protein G–Sepharose respectively. The beads were washed and bound proteins released from the antibodies with 0.1% SDS, subjected to SDS/PAGE and immunoblotted with anti-FLAG to detect Tpl2 and anti-HA to detect ABIN2. The immunoprecipitated Tpl2 was also immunoblotted with the antibodies described in the legend to Figure 5 to detect phosphorylation at Ser62, Thr290 and Ser400. (B) The experiment was carried out as described in (A) except that only wild-type Tpl2L was used and PP2 replaced C-1.

IL-1 triggers the dissociation of Tpl2 and ABIN2 in transfected IL-1R cells

The activation of Tpl2 is associated with its release from ABIN2, as well as from p105 [10]. To investigate whether the IL-1stimulated dissociation of Tpl2 from ABIN2 was regulated independently of the phosphorylation of p105, we co-transfected DNA vectors encoding both ABIN2 and the Tpl2 catalytic subunit into IRAK1-null IL-1R cells followed by stimulation with IL-1. These experiments showed that IL-1 induced the dissociation of Tpl2L from ABIN2 (Figure 9). Prior incubation of the cells with C-1 (Figure 9A) or PP2 (Figure 9B) did not prevent dissociation, indicating that the dissociation of these proteins was independent of Tpl2 activity or its activation. Similar results were obtained when Tpl2T was used instead of Tpl2L (results not shown).

DISCUSSION

It is well established that the activation of Tpl2 in macrophages by LPS or TNFα requires the IKKβ-catalysed phosphorylation of the p105 regulatory subunit of the Tpl2 complex, and that it is accompanied by the release of the Tpl2 catalytic subunit from both p105 and ABIN2 [7,13]. In the present study, we show that the activation of Tpl2 by IL-1 in IL-1R cells also requires the activation of IKKβ, because it is blocked by the IKKβ inhibitor PS1145 (Figures 1B and 1C). However, the IKKβ-catalysed phosphorylation of p105 is not sufficient for activation, and another signalling pathway is needed to activate the Tpl2 catalytic subunit. This second pathway does not require the presence of the IRAK1 protein or the IKKβ-catalysed phosphorylation of p105 (Figure 2 and Supplementary Figure S1), but is suppressed by PP2, a compound that does not affect IKKβ activity or the phosphorylation of p105 (Figure 4). This pathway appears to be physiologically relevant because the IL-1-stimulated activation of the endogenous Tpl2 complex in IRAK1-expressing IL-1R cells (Figure 3A), like the activation of the transfected catalytic subunit in IRAK1-null cells (Figure 4), is suppressed by PP2. Moreover, we have found that IL-1 induces the dissociation of the Tpl2 catalytic subunit from co-transfected ABIN2 and that this is also independent of IRAK1 (Figure 9) or the activation of IKKβ. However, in contrast with the activation of the Tpl2 catalytic subunit, the IL-1-induced dissociation of Tpl2 from ABIN2 is not suppressed by PP2, and it is also independent of the activation or activity of Tpl2. Our results suggest that three different signalling events are involved in the release and activation of the Tpl2 catalytic subunit (Figure 10). Whether these events occur in the sequence depicted in Figure 10 or in a different order is unknown at the present time.

The signalling pathways involved in the IL-1-stimulated activation of the Tpl2 complex

Figure 10
The signalling pathways involved in the IL-1-stimulated activation of the Tpl2 complex

The native form of Tpl2 in unstimulated cells is an inactive heterotrimer comprising the catalytic subunit complexed with ABIN2 and p105. Its activation requires IRAK1 and the PS1145-sensitive IKKβ-catalysed phosphorylation of p105, which leads to the release of p105 from Tpl2. IL-1 also induces the dissociation of ABIN2 and activates the Tpl2 catalytic subunit by pathways that do not require IRAK1 or the IKKβ-catalysed phosphorylation of p105. The IL-1-stimulated activation of the endogenous Tpl2 in IL-1R cells or the transfected Tpl2 catalytic subunit in IRAK1-null cells is suppressed by PP2, a compound that does not affect the activation of IKKβ or the phosphorylation of p105. The PP2-sensitive component is not a member of the Src family of protein tyrosine kinases, but may be another protein kinase designated here as protein kinase X (PKX). The IL-1-stimulated conversion of the Tpl2 catalytic subunit into an active conformation is followed by its autophorylation at Ser62, Thr290 and Ser400. The autophosphorylation of Thr290 appears to be required for Tpl2 to phosphorylate and activate MKK1. For simplicity, the three signalling pathways involved in the activation of the Tpl2 complex are depicted as occurring sequentially, but do not necessarily takes place in this order in vivo.

Figure 10
The signalling pathways involved in the IL-1-stimulated activation of the Tpl2 complex

The native form of Tpl2 in unstimulated cells is an inactive heterotrimer comprising the catalytic subunit complexed with ABIN2 and p105. Its activation requires IRAK1 and the PS1145-sensitive IKKβ-catalysed phosphorylation of p105, which leads to the release of p105 from Tpl2. IL-1 also induces the dissociation of ABIN2 and activates the Tpl2 catalytic subunit by pathways that do not require IRAK1 or the IKKβ-catalysed phosphorylation of p105. The IL-1-stimulated activation of the endogenous Tpl2 in IL-1R cells or the transfected Tpl2 catalytic subunit in IRAK1-null cells is suppressed by PP2, a compound that does not affect the activation of IKKβ or the phosphorylation of p105. The PP2-sensitive component is not a member of the Src family of protein tyrosine kinases, but may be another protein kinase designated here as protein kinase X (PKX). The IL-1-stimulated conversion of the Tpl2 catalytic subunit into an active conformation is followed by its autophorylation at Ser62, Thr290 and Ser400. The autophosphorylation of Thr290 appears to be required for Tpl2 to phosphorylate and activate MKK1. For simplicity, the three signalling pathways involved in the activation of the Tpl2 complex are depicted as occurring sequentially, but do not necessarily takes place in this order in vivo.

We studied the ability of IL-1 to activate different forms of the Tpl2 catalytic subunit following their transfection into IRAK1-null IL-1R cells. These experiments revealed that IL-1 activated Tpl2L and Tpl2S (the forms of Tpl2 that are present in vivo), as well as the C-terminally truncated species Tpl2T, but at different rates (Supplementary Figure S1). Tpl2L was activated most rapidly, consistent with earlier reports that the activation of this species underlies the initial rapid activation of ERK1/ERK2 in cells [7,13]. Interestingly, the activation of Tpl2S was much slower than Tpl2L, raising the possibility that it might play a role in maintaining a low level of activation of ERK1/ERK2 after Tpl2L has been inactivated by proteasomal destruction [7,13].

PP2 was originally developed as an inhibitor of the Src family of protein tyrosine kinases, but the concentrations of PP2 needed to prevent the IL-1-stimulated activation of Tpl2 were much higher than those required to suppress phosphorylation of p130CAS, a known physiological substrate of Src (Figure 3). Moreover SU6656, a structurally unrelated Src inhibitor suppressed the phosphoryation of p130CAS without affecting the IL-1-stimulated activation of Tpl2 (Supplementary Figure S2). Thus the involvement of Src family kinases in the activation of the Tpl2 catalytic subunit is excluded, suggesting that PP2 might exert its effect by inhibiting another protein kinase. PP2 does not inhibit Tpl2 activity in vitro, but does inhibits some other protein tyrosine kinases, such as the Bruton's tyrosine kinase and the Ephrin receptors A2 and B3 (J. Bain, M. Elliott and P. Cohen, unpublished work) and serine/threonine-specific protein kinases, such as CK1δ [27] and RIP2 (receptor-interacting protein 2) [27]. However, the possibility that PP2 might exert its effect by binding to a non-kinase target cannot be excluded at this stage.

The IL-1-stimulated activation of transfected Tpl2 in IRAK1-null cells was accompanied by the phosphorylation of Thr290 and Ser400 and the enhanced phosphoryation of Ser62 (Figure 5). Under these conditions, the phosphorylation of these sites appears to result from autophosphorylation catalysed by Tpl2 itself, because two different catalytically inactive mutants did not become phosphorylated in response to IL-1 (Figure 6). Moreover, Tpl2 that had been immunoprecipitated from IL-1-stimulated cells was able to phosphorylate all three sites in vitro (Figure 7A), and the phosphorylation of these sites was prevented by the Tpl2 inhibitor C-1. We have found that C-1 inhibits Tpl2 with an IC50 of 40 nM in vitro, but at a concentration of 1 μM has little effect on 70 other protein kinases tested (J. Moran and P. Cohen, unpublished work). These studies indicate that C-1 is a relatively selective inhibitor of Tpl2, although we cannot exclude the possibility that it inhibits other protein kinases that have not yet been tested.

Our mutagenesis studies demonstrated that the mutation of Ser62, Thr290 or Ser400 does not prevent the IL-1-stimulated conformational change that activates the Tpl2 catalytic subunit, because the singly mutated species are still capable of autophosphorylation (Figure 7B). However, the mutation of Thr290 to alanine, or the combined mutation of Ser62 and Ser400 to alanine, which prevents the phosphorylation of Thr290, does prevent Tpl2 from activating MKK1 (Figure 8A). These results suggest that the autophosphorylation of Tpl2 at Thr290 is required for the activation of MKK1, although not for the autophosphorylation of Ser62 and Ser400 (Figure 8B). The three-dimensional structure of Tpl2 has not yet been solved, but the region of the ‘activation loop’ in which Thr290 is located is known to be important for the interaction of other protein kinases with their substrates, such as PKB (protein kinase B) [28]. Alignment of the activation segment of Tpl2 with that of PKBβ shows that they are very similar, with Thr290 of Tpl2 aligning with Thr313 of PKB. Thr313 is very close to where a peptide corresponding to the phosphorylation site on GSK3β (glycogen synthase kinase 3β) binds to PKB, and could account for how the phosphorylation of Thr290 enables Tpl2 to activate MKK1 (D. Barford, personal communication).

In summary, the results of the present study support the view that the IL-1-stimulated phosphorylation of Tpl2 at Thr290 and Ser400, and its enhanced phosphorylation of Ser62 are autophosphorylation events that are a consequence of the IL-1-stimulated conversion of the Tpl2 catalytic subunit into an active conformation, the autophosphorylation of Thr290 probably being required for Tpl2 to interact with and phosphorylate MKK1 (Figure 10). However, further work is clearly needed to elucidate how IL-1 converts the catalytic subunit into the active conformation that is capable of autophosphorylation, which is independent of Thr290 phosphorylation.

Our finding that catalytically inactive mutants of Tpl2 do not become phosphorylated at Thr290 in response to IL-1 differs from earlier studies in which the catalytically inactive mutant Tpl2[K167M] was reported to become phosphorylated at Thr290 in transfected HEK-293 cells [16,17]. The reasons for the discrepancy with our results are unclear. It has also been reported by others that the transfection of DNA encoding Tpl2[S400A] into Tpl2−/− macrophages did not restore the LPS-stimulated activation of ERK1/ERK2 to these cells [15], suggesting a key role for Ser400 in the activation of the native Tpl2 complex. For example, it may facilitate the dissociation of Tpl2 from p105. It was also reported that LPS stimulates the phosphorylation of the catalytically inactive Tpl2L[D270A] at Ser400 in RAW mouse macrophages [15], suggesting that an LPS-stimulated Ser400 kinase, distinct from Tpl2, phosphorylates this site. This contrasts with the present study in which this catalytically inactive mutant did not become phosphorylated at Ser400 when transfected IRAK1-null cells were stimulated by IL-1 (Figure 6). The apparent discrepancy between these two studies could be explained if the LPS-stimulated phosphorylation of Tpl2[D270A] at Ser400 was catalysed by the endogenous Tpl2 catalytic subunit in transfected RAW cells, which would be present and activated under these conditions. Alternatively, LPS may activate a Ser400 kinase in RAW macrophages that is not expressed or not activated by IL-1 in IL-1R cells.

We thank Xiaolia Li and George Stark for generously providing the IRAK1−/− cells, David Barford for helpful discussions, Gursant Kular for the expression and purification of IL-1β, Natalia Shpiro for synthesizing C-1, and the antibody production team in the Division of Signal Transduction Therapy at Dundee for making the Tpl2 antibodies used in the present study.

Abbreviations

     
  • ABIN2

    A20-binding inhibitor of NF-κB 2

  •  
  • C-1

    Compound-1

  •  
  • DMEM

    Dulbecco's modified Eagle's medium

  •  
  • ERK

    extracellular-signal-regulated kinase

  •  
  • FCS

    foetal calf serum

  •  
  • GST

    glutathione transferase

  •  
  • HA

    haemagglutinin

  •  
  • HEK

    human embryonic kidney

  •  
  • IκB

    inhibitor of NF-κB

  •  
  • IKK

    IκB kinase

  •  
  • IL-1

    interleukin-1

  •  
  • IL-1R

    IL-1 receptor

  •  
  • IRAK1

    IL-1R-associated kinase

  •  
  • JNK

    c-Jun N-terminal kinase

  •  
  • LPS

    lipopolysaccharide

  •  
  • MAPK

    mitogen-activated protein kinase

  •  
  • MEF

    mouse embryonic fibroblast

  •  
  • MKK

    MAPK kinase

  •  
  • NF-κB

    nuclear factor κB

  •  
  • PKB

    protein kinase B

  •  
  • TNFα

    tumour necrosis factor α

  •  
  • Tpl2

    tumour progression locus 2

  •  
  • Tpl2L

    long form of Tpl2

  •  
  • Tpl2S

    short form of Tpl2

  •  
  • Tpl2T

    truncated form of Tpl2

AUTHOR CONTRIBUTION

Hosea Handoyo, Margaret Stafford and Philip Cohen designed the research; Hosea Handoyo, Margaret Stafford, Eamon McManus and Dionissios Baltzis performed the research; Mark Peggie contributed reagents; Philip Cohen and Hosea Handoyo wrote the paper.

FUNDING

This work was supported by Boehringer Ingelheim Fonds (postgraduate fellowship to H. H.); The Royal Society (a Royal Society Research Professorship to P. C.); the Medical Research Council; AstraZeneca; Boehringer Ingelheim; GlaxoSmithKline; Merck-Serono; and Pfizer.

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Author notes

1

These authors contributed equally to the present study.

Supplementary data