Within innate immune signaling pathways, divergent stimuli converge onto the IKK signalosome. Upon activation, IKKβ activates NF-κB transcription factors via the phosphorylation and proteasomal degradation of IκBα (reviewed in reference 11
). Little is known about the preferred substrate specificity of IKKβ, and few in vivo substrates have been identified. To address this limitation, we undertook a combined proteomic and bioinformatic approach to perform an unbiased search for novel IKKβ substrates. First, a positional scanning peptide library technique that we recently developed (14
) was used to identify the optimal phosphorylation motif for IKKβ. This technique utilizes a set of biotinylated peptide libraries, each of which contains a 50-50 mixture of serine and threonine fixed at the central position. In each library, one position relative to the central Ser/Thr is fixed with one of the 20 amino acids or with phosphothreonine (included to facilitate the identification of kinases which require priming by prior phosphorylation events). All other positions in each library contain an equimolar mixture of natural amino acids (excluding serine, threonine, and cysteine). Recombinant GST-IKKβ was purified from transfected HEK293T cells, and kinase assays were carried out on all 189 libraries simultaneously in solution, using [γ-32
P]ATP. Biotinylated peptides were captured using a streptavidin-coated membrane. For each position relative to the phosphorylation site, the relative amino acid preference was determined by the relative level of 32
P radiolabel incorporation into the corresponding peptide.
Using this method, we found that IKKβ exhibited significant sequence specificity at multiple positions relative to the phosphorylation site. The kinase strongly prefers peptides that contain phosphothreonine (and, presumably, phosphoserine) at the −4 and −5 positions relative to the phosphorylation site (Fig. ). Strong selectivity was also observed for aromatic residues at the −2 position, for hydrophobic residues at the +1 position, and for acidic residues at the +3 position (Fig. ). A kinase-deficient mutant preparation of IKKβ K44A did not phosphorylate any of the peptides, indicating that these results were not due to a contaminating kinase present in the IKKβ kinase preparation (Fig. ). This motif correlates well with several known IKKβ phosphorylation sites, including Ser32 and Ser36 of IκBα (DDRHDS32
GLDS and DpSGLDS36
MKDE, respectively) as well as Ser536 of the NF-κB transcription factor p65 (DEDFSS536
FIG. 1. Determination of preferred phosphorylation motif of IKKβ. (A and B) Recombinant IKKβ or, as a control, kinase-dead IKKβ K44A was used to phosphorylate 189 peptide libraries in individual kinase assays. The general sequence for (more ...)
Bioinformatic approaches were then used to identify candidate IKKβ substrates. First, spot intensities from the peptide library screen were quantified and converted into a matrix that could be used within the signaling search engine Scansite (data not shown). Scansite (http://scansite.mit.edu
) allows proteome-wide searches as well as searches within individual proteins for residues which best match the data provided by the matrix (22
). Proteome-wide searches frequently yield more candidates than can reasonably be tested, and results include numerous false-positive data (J. E. Hutti et al., unpublished observations). Because of this, we also used the IKKβ matrix to individually search proteins known to be involved in innate immune signaling for sequences that match the IKKβ phosphorylation motif. Once a list of candidate phosphorylation sites was identified, NetPhos (http://www.cbs.dtu.dk/services/NetPhos/
) was used to determine whether these sites are likely to be surface exposed and thus potentially phosphorylated in vivo (4
). Finally, the evolutionary conservation of candidate sites, in the context of conserved surrounding residues consistent with the optimal motif of IKKβ, was considered in determining the most likely candidate substrates. For instance, potential phosphorylation sites had to be conserved among all sequenced mammalian genomes and, preferably, among those of lower organisms.
Among the many candidate phosphorylation sites analyzed, Ser381 of A20 (TNFAIP3) emerged as a likely IKKβ phosphorylation site. A20 is a known negative regulator of NF-κB activity and has both K63 deubiquitinase and K48 E3 ligase activities (35
). Ser381 of this enzyme received a Scansite score comparable to those of known IKKβ phosphorylation sites, and NetPhos predicts that in vivo phosphorylation of Ser381 is very likely (Table ). In addition, Ser381, as well as the +1 leucine and +3 acidic residues, is evolutionarily conserved in mammals, in Xenopus laevis
, and in Danio rerio
, the lowest organism in which A20 is found (Fig. ).
Scansite and NetPhos scores for IKKβ substratesa
In order to verify that A20 could be phosphorylated directly by IKKβ, Flag-tagged A20 immunoprecipitated from HEK293T cells was incubated with purified GST-IKKβ and [γ-32P]ATP. We found that IKKβ could phosphorylate A20 in vitro and that phosphorylation was blocked by pretreatment with the IKKβ-specific inhibitor BMS-345541 (Fig. ). To identify the residue(s) on A20 that is phosphorylated by IKKβ, GST-IKKβ was cotransfected with Flag-A20 into HEK293T cells. Flag-A20 immunoprecipitates were subjected to stringent washing (1% NP-40, 0.25% deoxycholic acid, 1 M NaCl), followed by SDS-polyacrylamide gel electrophoresis and Coomassie staining (Fig. ). The stained band corresponding to A20 was subjected to tryptic digestion followed by reversed-phase microcapillary LC/MS/MS. This analysis identified phosphorylation of A20 at Ser381 (Fig. ), the same site that was predicted by our bioinformatic analysis.
FIG. 2. IKKβ phosphorylates A20 on serine 381 in vitro. (A) Flag-A20 was transfected into HEK293T cells and immunoprecipitated using an anti-Flag antibody. Immunoprecipitates were incubated with purified GST-IKKβ in the presence of BMS-345541 (more ...)
To determine whether this phosphorylation event occurs in vivo, we generated a phospho-specific antibody against this site. The specificity of the antibody was determined by cotransfecting GST-IKKβ with Flag-A20 or the point mutant Flag-A20 S381A. Flag immunoprecipitates were subjected to immunoblotting with the phospho-specific antibody. The antibody recognized wild-type (WT) A20 coexpressed with IKKβ but did not recognize the A20 S381A mutant (Fig. ). In addition, the phospho-A20 S381 antibody did not recognize A20 that had been treated with calf intestinal phosphatase, confirming that the antibody is specific for phosphorylated A20 (Fig. ).
FIG. 3. Anti-phospho-A20 S381 antibody recognizes A20 which is phosphorylated by IKKβ. (A) A phospho-specific antibody directed against A20 phospho-S381 was generated. To show specificity, Flag-A20 was cotransfected into HEK293T cells with GST-IKKβ (more ...)
Although these data strongly suggested that IKKβ phosphorylates A20 at S381, it was still possible that phosphorylation of A20 S381 is not direct. To address this possibility, a second in vitro kinase assay was performed. In this experiment, GST-IKKβ and Flag-A20 were cotransfected into HEK293T cells. Three hours prior to lysis, cells were treated with BMS-345541 to allow dephosphorylation of A20 in the absence of IKKβ activity. Following lysis, Flag-A20 was immunoprecipitated, and the immunoprecipitates were washed twice with lysis buffer and twice with kinase buffer. Immunoprecipitates were then resuspended in kinase buffer and preincubated for 20 min with BMS-345541 or vehicle control. ATP was then added to each reaction mix. Because no exogenous IKKβ was added following immunoprecipitation, all A20 phosphorylation occurring during this assay must be due to coprecipitating IKKβ. In accordance with this, coimmunoprecipitation experiments indicated that kinase-dead IKKβ can interact with A20 (data not shown). Following completion of the kinase reaction, the reaction mixes were immunoblotted using the phospho-A20 S381 antibody. Under these conditions, A20 was phosphorylated at position S381 and this phosphorylation was blocked by the IKKβ inhibitor BMS-345541, indicating that the kinase that phosphorylates this site coimmunoprecipitates with A20 and is inhibited by BMS-345541 (Fig. ). As a last test to determine the IKK dependence of this phosphorylation, NEMO+/+
MEFs were transduced with a retrovirus designed to express WT A20. After puromycin selection, clones (>1,000) were pooled and exposed to TNF-α. Lysates were generated, and A20 was immunoprecipitated through its Flag tag. Western blotting showed that in NEMO+/+
MEFs, S381 of A20 was phosphorylated, while in NEMO−/−
MEFs (with no TNF-induced IKK activity [reviewed in references 11
]), the S381A position of A20 was not phosphorylated (Fig. ). The results shown in Fig. , coupled with the results from Fig. , argue that IKKβ can directly phosphorylate A20 at S381.
Because A20 is a negative regulator of the NF-κB pathway (5
) and because A20−/−
mice are characterized by extreme hypersensitivity to LPS and TNF-α (18
), we hypothesized that A20 phosphorylation might be involved in the immune response to these stimuli. To test this possibility, partially differentiated THP-1 monocytes were pretreated for 3 h with BMS-345541 or vehicle control and then stimulated with LPS for 0 to 120 min. Endogenous A20 was immunoprecipitated, and the immunoprecipitates were immunoblotted using the phospho-A20 S381 antibody (Fig. ). While a strong signal was observed with the phospho-A20 S381 antibody at 90 and 120 min, inhibiting endogenous IKKβ activity with BMS-345541 pretreatment prevented a significant portion of this phosphorylation. To verify these results in a separate LPS-responsive cell line, mIMCD3 cells were transduced with a retrovirus expressing Flag-A20 or Flag-A20 S381A or with a vector control, and stable pools of A20-expressing cells were selected. Cells were stimulated with LPS for 0 to 60 min. Following LPS stimulation, Flag-A20 was immunoprecipitated, and immunoprecipitates were again immunoblotted using the phospho-A20 S381 antibody. In cells transduced with WT A20, a strong signal was observed with the phospho-specific antibody. However, this signal was not observed in cells expressing A20 S381A or the vector control (Fig. ). A similar result was obtained following stimulation of the same A20-expressing mIMCD3 cells with TNF-α (Fig. ). Together, these data show that A20 is phosphorylated at S381 in multiple cell types (IMCD cells are epithelial, THP-1 cells are hematopoietic, and MEFs are mesenchymal) following stimulation with IKKβ agonists.
FIG. 4. IKKβ phosphorylates A20 S381 in a stimulus- and time-dependent manner. (A) To induce expression of endogenous A20, THP-1 cells were differentiated for 16 h with 20 nM phorbol myristate acetate. These partially differentiated THP-1 cells were pretreated (more ...)
The phosphorylation of A20 by IKKβ could either enhance or inhibit the ability of A20 to downregulate NF-κB. To address this question, we first performed a luciferase reporter assay using an NF-κB-driven promoter transfected with proteins known to strongly induce K63-linked polyubiquitination of components of the NF-κB pathway (RIP2, NOD2, and TRAF6) (1
). While overexpression of these proteins gave various degrees of NF-κB activation, the NF-κB activity driven by either RIP2, NOD2, or TRAF6 was almost completely abrogated by coexpression of A20 (Fig. ). However, cotransfection of A20 S381A led to dramatically increased NF-κB reporter activation compared to that by WT A20 (Fig. ). This observation suggested that IKKβ phosphorylation of A20 at S381 results in increased A20 activity, which might provide a feedback mechanism to prevent uncontrolled inflammation.
FIG. 5. Phosphorylation of A20 S381 increases the ability of A20 to inhibit the NF-κB response. (A) HEK293T cells were transfected with an NF-κB-luciferase reporter gene, cytomegalovirus-driven Renilla luciferase (to standardize transfection efficiency), (more ...)
Activation of the Crohn's disease susceptibility complex, NOD2/RIP2, or the Toll-like receptor-activated E3 ubiquitin ligase TRAF6 leads to the K63-linked ubiquitination of NEMO at K285 (1
), and these K63-linked ubiquitin chains play an important role in coordinating innate immune signaling pathways (6
). Consistent with this, K63-linked ubiquitination of NEMO K285 is known to be essential for full IKKβ activity (1
). Since A20 is a K63 deubiquitinase, we decided to test whether A20 could suppress ubiquitination of NEMO. HA-ubiquitin, Myc-NEMO K399R, and OMNI-RIP2 were cotransfected into HEK293T cells, and the resulting NEMO ubiquitination produced was consistent with previously published data (1
). However, the addition of increasing amounts of A20 resulted in decreased RIP2-induced NEMO ubiquitination (Fig. ). NEMO K399R was used because elimination of the background ubiquitination at K399 facilitates the observation of NEMO K285 ubiquitination (1
), and these immunoprecipitations were all performed in high-stringency wash buffer (1 M NaCl). In addition, denaturing the lysate prior to immunoprecipitation to eliminate NEMO-binding proteins from the immunoprecipitates gave similar results (data not shown), indicating that the ubiquitination witnessed was due to NEMO ubiquitination and not to a coprecipitating protein. If phosphorylation of A20 by IKKβ increases A20 activity, then inhibition of IKKβ would lead to decreased A20 activity. This hypothesis was tested by observing decreases in the level of RIP2-induced NEMO ubiquitination as a readout for A20 activity. HA-ubiquitin, OMNI-RIP2, and Flag-A20 were cotransfected into 293T cells with Myc-NEMO K399R. Cotransfection of Myc-NEMO K399R with OMNI-RIP2 induced the expected increase in ubiquitination of NEMO at K285, and the addition of Flag-A20 eliminated a large portion of this ubiquitination (Fig. ). However, pretreating cells with BMS-345541 prior to lysis caused a large increase in the amount of NEMO ubiquitination, even in the presence of A20. Under these conditions, IKKβ activity was strongly inhibited by BMS-345541, as measured by IκBα degradation (data not shown). Consistent with the findings shown in Fig. , when Flag-A20 S381A replaced WT Flag-A20, the amount of NEMO ubiquitination also increased dramatically (Fig. ). Collectively, these findings support the hypothesis that IKKβ activity and phosphorylation of A20 at Ser 381 are required for full A20 activity.
In order to determine whether phosphorylation of A20 at S381 results in altered signaling following inflammatory stimuli in a more endogenous setting, A20−/−
MEFs were reconstituted with Flag-A20 WT, Flag-A20 S381A, or empty vector control. As described by Werner et al. (34
), reconstituted MEFs were pulsed with a nonsaturating level (2 ng/ml) of TNF-α for 30 min, and then the TNF-α-containing growth medium was replaced with serum-free medium after extensive phosphate-buffered saline (PBS) washing of the cells (Fig. ). By then assaying for recovery of IκBα via NF-κB-induced transcriptional activation, these conditions have been shown to assay the NF-κB-dependent negative regulation of TNF-induced signaling (34
). Cells were lysed every 15 min following removal of the TNF-α, and the recovery of IκBα levels was followed by immunoblotting. Between 30 and 75 min, we observed consistently higher levels of IκBα in cells reconstituted with WT A20 than in those with vector control (Fig. ). Previous studies comparing WT and A20−/−
MEFs have shown that this increase is due to the inhibition of NF-κB-activating pathways by A20 and therefore to a more rapid recovery of IκBα levels following TNF-α removal (18
). In our studies, the differences in IκBα levels observed between A20−/−
MEFs and A20-reconstituted MEFs are comparable to those observed previously using a similar time course (34
). Importantly, cells reconstituted with A20 S381A showed a time course of IκBα degradation and resynthesis matching that of vector-transduced cells, supporting the model that phosphorylation of A20 S381 is required for full A20 activity. To quantify this activity, the TNF-α stimulation was repeated for the 45-min time point in four separate experiments (Fig. ). At 45 min, the amount of IκBα present in the A20 S381A-transduced A20−/−
MEFs matched the amount in the vector-transduced cells and was significantly decreased relative to that in the WT A20-transduced MEFs. At a higher dose of TNF-α (6 ng/ml), this effect was even more pronounced, with very little IκBα produced in the A20-null MEFs reconstituted with A20 S381A (Fig. ). The findings presented in Fig. and strongly suggest that IKKβ phosphorylates A20 to increase its activity, thereby helping to limit the duration of NF-κB activity.
FIG. 6. Phosphorylation of A20 S381 leads to rapid inhibition of NF-κB following LPS stimulation. (A) A20−/− MEFs were stably reconstituted with Flag-A20 WT, Flag-A20 S381A, or vector control and pulsed with 2 ng/ml TNF-α for 30 (more ...)