Regulatory Serine Residues Mediate Phosphorylation-dependent and Phosphorylation-independent Activation of Interferon Regulatory Factor 7^*

Cell Culture

Human embryonic kidney 293T cells, monkey kidney COS cells, and murine TBK1-deficient immortalized embryo fibroblasts (a kind gift from W.-C. Yeh) were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% calf serum.

Transfections, Luciferase Assays, and Viral Infections

DNA transfections of 293T and COS cells were carried out by using standard calcium phosphate coprecipitation and DEAE-dextran methods, respectively. Transfection of TBK1-deficient fibroblasts was performed using Lipofectamine 2000 (Invitrogen). Luciferase activities were measured in cell lysates using commercial reagents as recommended by the manufacturer (Promega) and were normalized to β-galactosidase activity of a cotransfected CMV-lacZ plasmid measured on a luminescent substrate (Tropix). Each construct was tested in duplicate in at least three independent experiments. Results shown are from a single experiment representative of results obtained. Newcastle disease virus (NDV), Manhattan strain, was grown in 10-day embryonated chicken eggs (a kind gift from A. Garcia-Sastre), and viral infections were performed as previously described (2).

Plasmid Constructs and Mutagenesis

FLAG-tagged full-length murine IRF7 cDNA cloned into pcDNA3 and IFNα6-luciferase constructs have been described previously (2). FLAG-tagged IKKε and FLAG-tagged IKKεK38A expression vectors were kind gifts from T. Maniatis (7), and the TBK1 expression vector was a kind gift from D. Baltimore (6). The expression constructs encoding different IRF7 point mutants (M1–M18) were generated by site-directed mutagenesis (Stratagene) by using Pfu Turbo polymerase and were confirmed by DNA sequencing.

In Vitro Kinase Assay

In vitro kinase assays for IKKε were performed after overexpression of the FLAG-tagged protein in 293T cells. After immunoprecipitation using anti-FLAG (M2) beads (Sigma), the recovered protein was incubated in 20 μl of kinase buffer (50 mm Hepes, pH 7.6, 50 mm NaCl, 10 mm -glycerophosphate, 1 mm MgCl₂, 20 mm β- glycerophosphate NaF, 1 mm dithiothreitol) in the presence of 10 μCi of [γ-³²P]ATP and 1 μl of WT or mutant peptide at a final concentration of 0, 50, 100, or 200 μm at 30 °C for 30 min and analyzed by 10% SDS-PAGE and quantification using a PhosphorImager. Peptides were custom synthesized at the Keck Foundation, Yale University, using standard Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry.

Two-dimensional Gel Analysis

Nuclear extracts for isoelectric focusing were prepared as described previously (11). Extracts were loaded onto acrylamide gel strips containing 15 m urea, 3.5% Nonidet P-40, and 8% Pharmalyte™ 5–8 ampholins (Amersham Biosciences) and run at 200 V for 15 min, 300 V for 30 min, and 400 V overnight, followed by 800 V for 1 h. Gel strips were then equilibrated in 125 mm Tris-HCl, pH 6,8, 1% SDS, and 20% glycerol for 1 h at room temperature prior to sealing on 7.5% SDS-PAGE for the second dimension electrophoresis. Gels were transferred to polyvinylidene difluoride membranes and processed by Western blotting using anti-FLAG (M2) antibodies.

Electrophoretic Mobility Shift Assay

Nuclear extracts of 293T transfected cells were prepared as previously described (11). The electrophoretic mobility shift assay was performed by incubating nuclear extracts of each sample (2 μg) with a ³²P-labeled double stranded oligonucleotide corresponding to the IFN-stimulated response element sequence derived from the ISG15 gene (12), as described previously (13).

IRF7 Is Phosphorylated on Multiple Serine Residues within Its Carboxyl-terminal Regulatory Domain

Virally induced phosphorylation is a prerequisite to initiate the presumed conformational changes responsible for unmasking the potent transactivation ability of IRF7. Previous work has mapped the phosphorylation events to a serine/threonine-rich domain in the carboxyl terminus of IRF7 and shown that a truncated mutant lacking the last 34 amino acids was unresponsive to viral infection (2, 3). Targeted phosphorylation was further confirmed by phospho-amino acid analysis and phospho-peptide mapping of tryptic digests of IRF7. Virus infection led to an increase in IRF7 phosphoserine and the appearance of a single predominant novel V8 phosphopeptide.² The 34-amino acid regulatory domain of IRF7 contains 8 serine residues that are potential phosphorylation sites (Fig. 1A). However, which of these residues becomes phosphorylated has not been determined.

One of the assays commonly used to monitor IRF7 phosphorylation status is the appearance of a slower migrating band on SDS-PAGE. Although sensitive to qualitative changes in phosphorylation, this method cannot distinguish among multiple potential sites of phosphorylation. An alternative technique to detect different post-translational variants of the same protein is two-dimensional gel analysis. As shown in Fig. 1B, WT IRF7 from uninfected cells can be resolved into three distinct electrophoretic subspecies. Although these different spots are very likely due to post-translational modifications, treatment with alkaline phosphatase did not affect their distribution, suggesting that phosphorylation is not the cause of the observed pattern. However, when a similar analysis was performed on extracts from NDV-infected cells, 4–5 additional spots were resolved. These additional isoforms of IRF7 were indeed due to phosphorylated species, because they disappeared after phosphatase treatment, collapsing into the basal pattern. This result shows that IRF7 can be phosphorylated at multiple distinct sites. It also indicates that virus infection does not achieve complete phosphorylation of the protein, leading to coexistence of several species bearing different degrees of phosphorylation.

Interestingly, overexpression of either TBK1 or IKKε, kinases responsible for IRF7 phosphorylation, led to complete phosphorylation, shown by almost total disappearance of the pre-existing spots (Fig. 1B). This result showed that each of the three pre-existing species could be phosphorylated and that action of either kinase was sufficient to produce the same bulk phosphorylation pattern. Notably, overexpression of both kinases together did not lead to further alteration of the two-dimensional pattern (data not shown).

Two Groups of Serine Are Necessary for Virally Induced Phosphorylation of IRF7

To determine the major targets of virally induced phosphorylation, we mutated each contiguous group of serine residues in the regulatory domain into alanine, a residue that cannot be phosphorylated. The status of phosphorylation of the different mutants was monitored by two-dimensional gel analysis before and after viral infection. This analysis revealed that loss of two distinct sets of serine residues, Ser⁴²⁵ and Ser⁴²⁶ and Ser⁴³⁷ and Ser⁴³⁸, rendered IRF7 unresponsive to viral infection. In contrast, mutation of the other two sets of serines, Ser⁴²⁹–Ser⁴³¹ and Ser⁴⁴¹, did not show a significant alteration in the overall phosphorylation pattern, although the efficiency of phosphorylation appeared to be reduced (Fig. 1C). We noted that leucine residues are associated with these serine residues, suggesting a potential phosphorylation motif. Specifically, each group of serine residues is either preceded or followed by a leucine residue. To address the importance of these leucine residues, we mutated individually Leu⁴²⁷ and Leu⁴³² into alanines. As shown in Fig. 1C, mutation of Leu⁴²⁷ abrogated virally induced phosphorylation, whereas mutant L432A showed the same phosphorylation pattern as WT IRF7. Notably, this result is consistent with what was observed when the adjacent serine sets were altered, in other words, it seems that the motif SSL (amino acids 425–427) has a preponderant role in virally induced phosphorylation, whereas the next motif SSSL (amino acids 429–432) has a minor role, if any.

Ser⁴³⁷ and Ser⁴³⁸ Are the Primary Targets of Phosphorylation

To better determine the respective roles of each group of serines, we undertook another series of mutations. The different sets of serine residues were altered to aspartate, a residue that mimics the charge of a phosphorylated serine and can often substitute for its function. Analysis by two-dimensional gel confirmed that these mutants had an altered charge. In addition, we observed that most mutants were still further phosphorylated in response to viral infection, as attested by the appearance of new spots toward the acidic end of the pH gradient (Fig. 1D). Interestingly, the pattern of mutant M7 (S425D/S426D) was not modified after viral infection suggesting that either this mutation rendered the protein unresponsive to viral infection or that these two serines were the only targets for phosphorylation. It is unlikely that Ser⁴²⁵ and Ser⁴²⁶ are the only sites of phosphorylation given the multiple isoelectric species detected after phosphorylation of the wild type protein (Fig. 1B), indicative of more than two phosphorylated residues. Moreover, mutant M15, where all the serines except Ser⁴²⁵ and Ser⁴²⁶ were mutated into aspartate, showed a dramatic shift toward the acidic pH, resembling the fully phosphorylated protein and an unchanged pattern when analyzed from NDV-infected extracts. One hypothesis that reconciles these observations is that the distal serine residues are major targets for virus-induced phosphorylation, whereas Ser⁴²⁵ and Ser⁴²⁶ are necessary for kinase targeting but are either not themselves phosphorylated or their function is not correctly mimicked by aspartate substitution.

Transcriptional Activity of IRF7 Mutants

Although informative, two-dimensional gel migration patterns were not sufficient to give an answer concerning the functionality of the different residues, especially considering the limitations of phosphomimetic mutants. To complement this study, it was essential to determine the level of activity of each mutant in control and infected cells. For this purpose, we cotransfected WT IRF7 and the different mutants in COS cells along with a luciferase reporter construct driven by the IFNα6 promoter, a typical target gene of IRF7 (2). As shown previously, WT IRF7 transactivation ability was greatly enhanced (over 8-fold) following viral infection (2). However, mutation of Ser⁴²⁵ and Ser⁴²⁶ or Ser⁴³⁷ and Ser⁴³⁸ totally abrogated virally induced activity (Fig. 2A). Moreover, mutant M2 (L427A) also showed a complete loss of viral responsiveness. The other mutants, where Ser⁴²⁹–Ser⁴³¹, Ser⁴⁴¹, or Leu⁴³² were mutated, were not significantly impaired in their viral sensitivity compared with WT. These results are fully consistent with what was observed with the phosphorylation status, reaffirming that virally induced phosphorylation is the key regulatory event to derepress transactivation ability.

A more complicated situation was observed when the activity of the phosphomimetic mutants was measured. Consistent with the finding that mutation of Ser⁴²⁹–Ser⁴³¹ to Ala did not have a noticeable effect on the virus-induced pattern or transactivation potential, mutation of the same residues into Asp, either individually or taken together (mutants M8, M9, and M10), did not render the mutants constitutively active. However, M10 reached a level of activity 4-fold higher than WT in the presence of virus (Fig. 2B). This acute sensitivity to viral infection suggests that these residues (Ser⁴²⁹–Ser⁴³¹) are not, or at most very inefficiently, phosphorylated in response to viral infection. However, if they were to become phosphorylated, as mimicked by the aspartate mutation, they could cooperate with the residues that are primary targets for the virus-activated kinases, yielding higher transactivation potential. Again, as predicted by the phenotype observed after mutation into alanine, M14 (S437D/S438D) showed a significant constitutive activity in absence of virus, 7-fold higher than the WT level, consistent with these residues being the major targets for virally induced phosphorylation. Interestingly, additional mutation of serine residues 429–431 (M11) rendered the mutant highly constitutively active, reaching levels of activity in the range of the wild type protein following viral infection. Furthermore, when we generated a mutant (M15) where Ser⁴⁴¹ was mutated into aspartate in addition to the five mutations present in M11, we obtained a protein that displayed even a greater activity, reaching levels 5-fold higher than WT IRF7 activated by viral infection. It is important to note that activity of both M11 and M15 was not further increased by viral infection, suggesting that no additional phosphorylation could take place. We conclude that phosphorylation of Ser⁴³⁷ and Ser⁴³⁸ is required for virus-induced transactivation, whereas phosphorylation of Ser⁴²⁹–Ser⁴³¹ and/or Ser⁴⁴¹ can augment transactivation and/or the efficiency of distal phosphorylation.

This notion raised the question of the physiological role of the first set of serines, Ser⁴²⁵ and Ser⁴²⁶, that were previously found to be essential to virally induced phosphorylation and activity (Ref. 2 and Figs. 1C and 2A). Surprisingly, when Ser⁴²⁵ and Ser⁴²⁶ were mutated into aspartate, the resulting mutant (M7) did not exhibit constitutive activity but rather became insensitive to viral infection. This phenotype is consistent with our previous finding that this mutant could not be phosphorylated in response to viral infection (Fig. 1D) and suggests that aspartate cannot mimic the essential role of these serine residues.

Transactivation ability is the ultimate indication of IRF7 activity. Nevertheless, lack of or increased activity of the mutants could be because of alteration of other parameters that are a prerequisite to IRF7 transactivation ability, namely dimerization, retention in the nucleus, or DNA binding. We therefore tested the DNA binding ability of several mutants. As previously described (14), DNA binding by WT IRF7 was dramatically increased in NDV-infected extracts. Interestingly, constitutively active mutants M14 and M15 showed highly increased DNA binding capabilities in control extracts compared with WT. Whereas the DNA binding ability of M14 was further enhanced after viral infection, M15 exhibited the same degree of DNA binding in control and NDV-infected extracts (Fig. 3). These findings are reminiscent of what was observed when we monitored IRF7 activity by luciferase assay, showing a strong correlation between stimulation of DNA binding and transcriptional activity.

Essential Functions of Ser⁴²⁵ and Ser⁴²⁶

The fact that the mutation of Ser⁴²⁵ and Ser⁴²⁶ to aspartate did not confer increased activity suggested that these residues might not be targets of phosphorylation, although they played an essential role in IRF7 function. We considered whether their critical role impacted proper conformation of the regulatory domain or served as a recognition site for the virally activated kinases, IKK-ε and TBK1.

To test this hypothesis, we mutated the proximal group of serines, Ser⁴²⁵ and Ser⁴²⁶ either to alanine or aspartate, in the context of the constitutively active phosphomimetic mutants M11 and M15. We reasoned that, if the only function of the proximal set of serines was to provide a docking site for the kinases that will subsequently phosphorylate the distal group of serine (437 and 438), their mutation in the context of constitutively active mutants would have a limited impact. Unexpectedly, as shown in Fig. 4A, these mutations partially impaired the activity of the constitutively active mutants (compare mutant M12 with M11 and mutants M16 and M17 with M15). These results indicated that Ser⁴²⁵ and Ser⁴²⁶ were critical, even when all other serine residues in the regulatory domain available for phosphorylation were already mutated into aspartate, suggesting either that these serines are obligatory phosphorylation targets or that they are mandatory for adequate folding of the carboxyl-terminal transactivation domain. To determine whether these residues were phosphorylated in the context of M15, we compared the two-dimensional pattern of M15 with M17 where Ser⁴²⁵ and Ser⁴²⁶ are mutated into alanine. As shown in Fig. 4B, M17 lost the more acidic minor spot present in M15, suggesting that M15 may indeed be partially phosphorylated on residues Ser⁴²⁵ and Ser⁴²⁶. However, mutation of Ser⁴²⁵ and Ser⁴²⁶ into either Asp or Ala reduced but did not abrogate the constitutive activity of mutant M11 and M15. Mutants M12 and M17 still exhibited a transactivation ability 5–8-fold higher than WT IRF7 in control cells, reaching levels of activation close to that of viral activated WT IRF7 (Fig. 4A). This result indicates that although Ser⁴²⁵ and Ser⁴²⁶ are important to achieve maximal activity, these residues are not compulsory to obtain a constitutively active mutant.

Nevertheless, this result did not definitively exclude the possibility that these residues also serve as a recognition motif in the context of the WT protein. To address this point, we designed a synthetic peptide encompassing the sequence surrounding the proximal SSL motif, i.e. amino acids 422–435, fused to the protein transduction domain (amino acid 47–57) of the human immunodeficiency virus type 1 transactivator protein Tat (15). As a control, we also synthesized a mutant peptide bearing the same sequence except that the SSL motif was mutated into triple alanine (Fig. 5A). The potential interaction between the peptides and the IRF7 kinase, IKK-ε, was tested by quantifying the level of IKK-ε autophosphorylation in an in vitro kinase assay in presence or absence of the WT or mutant peptide (Fig. 5B). WT peptide strongly inhibited IKK-ε autophosphorylation in a dose-dependent manner, suggesting that the kinase efficiently interacted with the peptide. The corresponding mutant peptide also showed some degree of inhibition, although this inhibition did not show a clear dose-response effect, suggesting that it was likely to be low affinity or nonspecific (Fig. 5B).

Mori et al. (16) recently proposed that constitutive activity of IRF3-5D (analogous to mutant M15 in this study) was because of the constitutive phosphorylation of the equivalent proximal set of serine residues (Ser³⁸⁵ and Ser³⁸⁶ in IRF3). To examine the possibility of a similar phenomenon in IRF7, we assayed IRF7 WT and M15 transactivation ability in the IFNα6-luciferase reporter assay using cells that could be depleted of the kinase activity responsible for phosphorylating IRF7, namely TBK1 and IKK-ε. For this purpose, we used TBK1-deficient fibroblasts from gene-targeted mice and transfected them with a dominant negative form of IKK-ε, IKK-εK38A (6), to impair its activity. In these cells virtually devoid of the relevant kinase activity capable of targeting IRF7 carboxyl-terminal regulatory domain, WT IRF7 lost its low basal activity, whereas M15 retained robust constitutive activity (Fig. 5C). As a control, TBK1-deficient cells reconstituted with TBK1 showed a high constitutive activity of WT IRF7, reaching levels ~1000-fold higher than in the absence of kinases. In contrast, M15 activity was only 4-fold greater in cells transfected with TBK1. This result strongly suggested that M15 activity was not because of constitutive phosphorylation of Ser⁴²⁵ and Ser⁴²⁶ by TBK1 or IKK-ε, whereas the basal activity of WT IRF7 is probably because of low levels of constitutive IKK catalytic activity.

In another tactic to determine whether Ser⁴²⁵ and Ser⁴²⁶ were targets of phosphorylation, we designed a mutant, M18, derived from the phosphomimetic mutant M14 (S437D/S438D). As discussed above, M14 is weakly constitutively active but still strongly activated by virus. Moreover, our results indicated that Ser⁴²⁹–Ser⁴³¹ and Ser⁴⁴¹ were not essential residues to achieve viral-induced phosphorylation (Figs. 1C and 2A, M4 and M6). A mutant combining the mutations of M14, M4, and M6 only leaves Ser⁴²⁵ and Ser⁴²⁶ as potential phosphorylation targets in response to viral infection. We tested the ability of such a mutant to be activated in response to viral infection. As shown in Fig. 5D and E, mutant M18 was not responsive to NDV infection, either by increased transcriptional activity or by altered electrophoretic mobility.

Interestingly, however, M18 resolved as a major isoelectric species plus a minor, more acidic species (Fig. 5E) that was lost when Ser⁴²⁵ and Ser⁴²⁶ were mutated to Ala in M17, suggesting a degree of phosphorylation (Fig. 4B). This result is consistent with the conclusion that Ser⁴²⁵ and Ser⁴²⁶ are not primary targets for virally induced phosphorylation, although they may become secondarily phosphorylated and then contribute to the overall phosphorylation-dependent activity of IRF7. Furthermore, although the groups of Ser⁴²⁹–Ser⁴³¹ and Ser⁴⁴¹ seem dispensable when mutated individually, the combined mutation of both sets rendered IRF7 totally inactive. This result indicates that these serine residues are redundant with each other but that the presence of at least one set, Ser⁴²⁹–Ser⁴³¹ or Ser⁴⁴¹, is crucial for viral responsiveness of IRF7.