The Major Sites of IKKβ-Mediated In Vitro Phosphorylation on REL Are Not Required for IKKβ-Enhanced Transactivation by GAL4-REL
Several groups have shown that sequences C-terminal to the RHD in REL can be phosphorylated by IKK in vitro; however, the individual phosphorylation sites have not been precisely identified. Namely, REL can be phosphorylated between residues 422 and 540 by IKKα and between residues 473 and 531 by IKKβ (4
). Starczynowski et al. (23
) showed that REL can be weakly phosphorylated by IKKβ on Ser525, but showed that there are additional sites of phosphorylation.
To identify the major residues in the C-terminal half of REL that are phosphorylated by IKK, immune complex kinase assays were performed using GST-REL fusion proteins containing aa 323–587 and a series of deletions of residues from the REL C-terminus (). FLAG-tagged IKKβ was immunoprecipitated from transfected A293 cells and incubated with these bacterially expressed GST-REL substrates. GST-REL aa 323–587, aa 323–529 (Δ58), and aa 323–497 (Δ90) were strongly phosphorylated by IKKβ, whereas GST-REL aa 323–477 (Δ110) and aa 323–455 (Δ132) were not appreciably phosphorylated above the level seen with GST alone. Coomassie blue staining shows approximately equal loading of all substrates. These results indicate that the major IKKβ phosphorylation sites on REL are between residues 477 and 497.
IKK is almost exclusively a serine-specific protein kinase (20
). Between aa 477 and 497, there are three serine residues, at residues 484, 491, and 494. To determine which specific sites are phosphorylated by IKKβ, immune complex kinase assays were performed using a GST-REL substrate containing aa 476–504 and single mutants S484A, S494A, or double mutant S484,494A (). The wild-type REL peptide and both single mutants were readily phosphorylated, whereas the GST-REL-S484,494A double mutant was not. This result shows that Ser484 and Ser494 are IKKβ phosphorylation sites in vitro and rules out Ser491 as a site of IKKβ phosphorylation.
An IKKβ immune complex kinase assay was next performed using these same mutants in the context of REL aa 323–587, containing the entire C-terminal transactivation domain of REL (). GST-REL and both single mutants (S484A and S494A) were phosphorylated strongly, whereas mutant S484,494A showed greatly reduced phosphorylation (GST-REL-Δ110 was included as a negative control). We conclude that Ser484 and Ser494 are the major sites of in vitro phosphorylation by IKKβ in the C-terminal half of REL. We have also determined by immune complex kinase assays that Ser484 and Ser494 are sites of IKKβ phosphorylation (data not shown).
To determine whether REL can be phosphorylated on Ser484 and Ser494 by endogenous IKK, kinases assays were performed using immunoprecipitated IKK complexes from 3T3 cells or from comparable IKKα−/−, IKKα−/−, and IKKα/IKKβ−/− mouse fibroblast cell lines. Cells were either untreated or were stimulated with TNF-α, and IKK complexes were immunoprecipitated with anti-NEMO antiserum. Immune complex kinase assays were then performed using GST, GST-REL, and GST-REL-S484,494A as substrates, either as contained within the aa 476–504 peptide () or within the entire C-terminal half of REL (aa 323–587) (). IKK complexes immunoprecipitated from 3T3 cells and both single IKK knockout cells phosphorylated wild-type GST-REL, but not GST alone. GST-REL-S484,494A was phosphorylated much more weakly than wild-type REL in the immune complexes from 3T3 and single IKK knockout cells (). GST-REL fusion proteins were not phosphorylated using IKK complexes immunoprecipitated from the IKKα/IKKα−/− double knockout cells. Therefore, endogenous IKKα and IKKβ appear to show the same specificity for phosphorylating REL Ser484 and Ser494 as overexpressed IKKα and IKKβ.
To determine whether IKKβ can phosphorylate REL on Ser484 and Ser494 in vivo, we performed 32P metabolic labeling. A293 cells were transfected with FLAG-tagged REL or REL-S484,494A, with and without HA-IKKβ-S177,181E (SS/EE), a constitutively active IKKβ mutant. Cells were radiolabeled with [32P]orthophosphate and FLAG-REL proteins were immunoprecipitated. In all cases, REL and REL-S484,494A showed similar amounts of phosphate labeling (). Anti-REL Western blotting confirmed that the radiolabeled bands contained approximately equal amounts of REL protein. Therefore, Ser484 and Ser494 in REL are unlikely to be sites of in vivo phosphorylation by IKKβ.
It has been shown previously that cotransfection with IKKβ can increase the transactivation ability of GAL4-REL about fourfold in A293 human embryonic kidney cells (23
). We repeated this assay in A293 cells, using IKKβ-SS/EE and the same GAL4-REL fusion protein containing the REL transactivation domain (aa 278–587) and measured transactivation from a GAL4 site luciferase reporter. GAL4-REL can activate reporter gene activity by over 200-fold compared to GAL4 (1–147) alone (21
); therefore, to achieve a maximal effect of IKKβ on GAL4-REL transactivation, we optimized the assay by titrating down the amount of GAL4-REL expression plasmid compared to the IKKβ-SS/EE expression plasmid (100 ng pSG-REL to 0.5 μg FLAG-IKKβ-SS/EE). Under these conditions, we observed an approximately 30-fold increase in GAL4-REL transactivation when cells were cotransfected with an expression plasmid for the constitutively active IKKβ-SS/EE protein, compared to cells cotransfected with the empty vector control ().
To determine whether IKK-mediated enhancement of GAL4-REL transactivation is due to phosphorylation of REL by IKK at serine residues 484 and 494, we measured transactivation by GAL4 fusion proteins containing the C-terminal sequences from wild-type REL or REL-S484,494A, with and without IKKβ-SS/EE in A293 cells (). In the absence of IKKβ-SS/EE, GAL4-REL and GAL4-REL-S484,494A activated transcription to approximately the same extent. Moreover, IKKβ-SS/EE increased transactivation by both GAL4-REL and GAL4-REL-S484,494A by approximately 30-fold (). In similar experiments with (wild-type) IKKα, both GAL4-REL and GAL4-REL-S484,494A activated transcription approximately threefold higher when cotransfected with IKKα than with the vector control. We have conducted similar experiments using full-length REL and REL S484,494A and a κB site reporter plasmid. We observed no significant differences in transactivation by wild-type REL versus REL-S484,494A (data not shown). Taken together, these results show that IKK can increase the transactivation ability of GAL4-REL, but that this effect does not require phosphorylation of Ser484 or Ser494, which are major sites of REL phosphorylation by IKK in vitro.
The IκBαSuper-Repressor Abolishes IKKβ- and NF-κB-Mediated Enhancement of GAL4-REL Transactivation
Because mutation of the major IKKβ phosphorylation sites in REL does not affect IKKβ-mediated enhancement of GAL4-REL transactivation, it suggested to us that this enhancement proceeds through a mechanism other than IKKβ phosphorylation of REL. As a first step towards describing this indirect mechanism, we sought to determine whether disruption of downstream effects of IKKβ on NF-κB affected its ability to enhance GAL4-REL transactivation. Because IKKβ-SS/EE is a potent inducer of NF-κB, we determined the effect of the IκBα super-repressor (IκBα-SR) on IKKβ enhancement of GAL4-REL transactivation. The IκBα-SR does not dissociate from NF-κB dimers, and therefore blocks activation of NF-κB by IKK. Expression of the IκBα-SR had no significant effect on transactivation by GAL4-REL in the absence of IKKβ-SS/EE (). However, cotransfection of the IκBα-SR blocked the ability of IKKβ-SS/EE to enhance transactivation by GAL4-REL (), suggesting that the effect of IKKβ-SS/EE on GAL4-REL transactivation requires increased NF-κB activity.
Figure 2 The IκBα super-repressor blocks IKKβ- and NF-κB-mediated enhancement of GAL4-REL transactivation. (A) Cells were transfected with 100 ng of pSG-REL expression vector, 0.5 μg pcDNA, IκBα-SR, or IKKβ-SS/EE, (more ...)
The cytokine TNF-α can activate the canonical NF-κB pathway, which proceeds through activation of the IKK complex (5
). TNF-α has also been shown to enhance GAL4-REL transactivation in cell-based GAL4-site reporter gene assays (13
). To determine whether the effect of TNF-α on GAL4-REL is due to IKK activation of NF-κB signaling, A293 cells were transfected with GAL4-REL and either vector alone or IκBα-SR. These cells were then either treated with TNF-α or left untreated. As previously observed (13
), TNF-α treatment increased GAL4-REL transactivation by approximately threefold compared to untreated cells (). However, expression of the IκBα-SR blocked the TNF-α-induced increase in GAL4-REL transactivation (). These data show that the IκBα-SR blocks the ability of both overexpressed IKKβ- and TNF-α-activated endogenous IKK to enhance GAL4-REL transactivation, suggesting that downstream activation of NF-κB is required for the effects of IKKβ on GAL4-REL.
To determine whether overexpression of NF-κB subunits could also enhance GAL4-REL transactivation, we cotransfected A293 cells with GAL4-REL, and with vector alone or expression plasmids for REL, RelA, or, as a positive control, IKKβ-SS/EE. As previously observed, IKKβ-SS/EE increased GAL4-REL by approximately 30-fold. REL and RelA increased GAL4-REL transactivation by approximately sixfold and eightfold, respectively (). Furthermore, the IκBα-SR blocked the ability of RelA to enhance GAL4-REL transactivation ().
Taken together, these results show that NF-κB activity increases GAL4-REL transactivation and suggest that induction of NF-κB signaling accounts for the effect of IKKβ on GAL4-REL transactivation in these types of reporter gene assays.
The pSG424 GAL4-Fusion Protein Expression Vector Contains κB-Responsive Sites in Its SV40 Promoter Sequences
Upon examination of the Simian virus 40 (SV40) early promoter sequences in the pSG424 vector, which is used for expression of GAL4 and GAL4-REL, we identified two κB sites (5′-GGAAAGTCCCC-3′) that have been previously identified by Kanno et al. (8
). To determine whether the reason for enhancement of GAL4-REL transactivation by NF-κB and IKKβ was due to NF-κB-mediated activation of the promoter in the pSG424 vector, we first subcloned the pSG424 SV40 promoter sequences into the luciferase reporter vector pGL3 Promoter Vector. Cotransfection of RelA with pSV40-luciferase resulted in an approximately 16-fold higher luciferase activity than the vector control. Additionally, cotransfection of IKKβ-SS/EE with pSV40-luciferase resulted in an approximately 55-fold higher luciferase activity than the vector control ().
Figure 3 The pSG424 vector contains κB-responsive sites in its SV40 promoter sequences. Cells were transfected with 1 μg pcDNA, RelA, or IKKβ-SS/EE, and either 0.5 μg of SV40-luciferase plasmid (SV40-luc) or 0.5 μg of RSV-luciferase (more ...)
The Rous sarcoma virus (RSV) promoter is not known to contain κB sites. To determine whether IKKβ and NF-κB could affect expression from the RSV promoter, we subcloned the RSV promoter into the pGL3 Promoter Vector. We cotransfected RelA and IKKβ-SS/EE with pRSV-luciferase and observed only minor increases (approximately twofold) in luciferase activity when compared with the vector control ().
These results suggest that pSG424 contains functional κB sites and that NF-κB increases expression from the promoter in pSG424, which accounts for the NF-κB- and IKKβ-mediated increases in GAL4-REL transactivation.
Replacement of the SV40 Promoter With the RSV Promoter in the pSG424 Vector Abolishes NF-κB- and IKKβ-Mediated Enhancement of GAL4-REL Transactivation
To show more directly that the κB site-containing promoter sequences in the pSG424 vector account for IKKβ-mediated induction of GAL4-REL, we replaced the SV40 promoter sequences in the pSG424 vector with the RSV promoter sequences to create the pRG424 vector, and then assessed whether IKKβ-SS/EE could affect GAL4-REL transactivation (). As a control, cotransfection of pSG-REL with IKKβ-SS/EE resulted in an approximately 15-fold increase in transactivation by GAL4-REL compared to cotransfection with the vector control. However, cotransfection of the IKKβ-SS/EE expression vector with pRG-REL or pRG-REL- S484,494A did not increase GAL4-REL transactivation above the level seen with the vector control (); in fact, we even noted a decrease in GAL4-REL transactivation in the presence of IKKβ-SS/EE. In addition, we observed only a minor difference in the basal transactivation by pRG-REL and pRG-REL S484,494A.
Figure 4 Replacement of the SV40 promoter with the RSV promoter in the pSG424 vector abolishes IKKβ-mediated enhancement of GAL4-REL transactivation. (A) Cells were transfected with 100 ng of pSG424 GAL4-fusion expression vectors, 10 ng of pRG424 GAL4-fusion (more ...)
To achieve high constitutive expression of GAL4-REL, we subcloned the GAL4-REL sequences into pcDNA 3.1. pcDNA 3.1 contains the cytomegalovirus (CMV) promoter, which is a much stronger promoter than the SV40 promoter (26
), presumably directing greater expression of GAL4-REL. Transfection of 100 ng of pcDNA-GAL4-REL resulted in an approximately 70-fold increase in reporter gene expression compared to 100 ng of pSG-REL (). Of note, the RSV promoter is also about 80-fold stronger than the SV40 promoter: that is, transfection of 10 ng of pRG-REL yields approximately eight times more GAL4-site promoter luciferase activity than transfection of 100 ng of pSG-REL (). Taken together, these results show that increased levels of promoter activity controlling GAL4-REL protein expression are correlated with increased expression of luciferase from the GAL4-site reporter plasmid.
To show that increased GAL4-REL protein expression also correlates with increased GAL4-site luciferase activity, we cotransfected A293 cells with pSG424, pSG-REL, pRG424, or pRG-REL and with pcDNA or pcDNA-IKKβ-SS/EE. Anti-REL Western blot analysis of nuclear extracts showed that cotransfection of IKKβ-SS/EE with pSG-REL results in increased expression of GAL4-REL protein (, lanes 3 and 4); in contrast, cotransfection of IKKβ-SS/EE did not appreciably affect the levels of GAL4-REL expressed from pRG-REL (, lanes 7 and 8). Additionally, pcDNA-GAL4-REL directs high levels of expression of GAL4-REL, similar to what is seen with pRG-REL alone and pSG-REL when cotransfected with IKKβ-SS/EE (, compare lanes 4, 7, and 9). We also show that TNF-α treatment of cells transfected with pSG-REL results in increased expression of GAL4-REL protein ().
These results show that cotransfection of the IKKβ-SS/EE expression vector and activation of endogenous IKK by TNF-α correlate with both increased GAL4-site luciferase reporter gene activity and increased levels of GAL4-REL protein when using the pSG424 vector, which contains functional κB sites. Conversely, cotransfection of the IKKβ-SS/EE expression plasmid does not result in increased GAL4 site luciferase reporter activity or GAL4-REL protein levels when using the pRG424 vector, which lacks known functional κB sites.