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

Abstract

Interferon regulatory factor (IRF)7 is a key transcription factor required for establishment of antiviral resistance. In response to infection, IRF7 is activated by phosphorylation through the action of the non-canonical IκB kinases, IκB kinase-ϵ and TANK-binding kinase 1. Activation leads to nuclear retention, DNA binding, and derepression of transactivation ability. Clusters of serine residues located in the carboxyl-terminal regulatory domain of IRF7 are putative targets of virus-activated kinases. However, the exact sites of phosphorylation have not yet been established. Here, we report a comprehensive structure-activity examination of potential IRF7 phosphorylation sites through analysis of mutant proteins in which specific serine residues were altered to alanine or aspartate. Phosphorylation patterns of these mutants were analyzed by two-dimensional gel electrophoresis, and their transcriptional activity was monitored by reporter assays. Essential phosphorylation events were mapped to amino acids 437–438 and a redundant set of sites at either amino acids 429–431 or 441. IRF7 recovered from infected cells was heterogeneously phosphorylated at these sites, and greater phosphorylation correlated with increased transactivation. Interestingly, a distinct serine cluster conserved in the related protein IRF3 was also essential for IRF7 activation and distal phosphorylation. However, the essential role of this motif did not appear to be fulfilled by phosphorylation. Rather, these serine residues and an adjacent leucine were required for phosphorylation at distal sites and may determine a conformational element required for function. Interferon regulatory factor (IRF)7 is a key transcription factor required for establishment of antiviral resistance. In response to infection, IRF7 is activated by phosphorylation through the action of the non-canonical IκB kinases, IκB kinase-ϵ and TANK-binding kinase 1. Activation leads to nuclear retention, DNA binding, and derepression of transactivation ability. Clusters of serine residues located in the carboxyl-terminal regulatory domain of IRF7 are putative targets of virus-activated kinases. However, the exact sites of phosphorylation have not yet been established. Here, we report a comprehensive structure-activity examination of potential IRF7 phosphorylation sites through analysis of mutant proteins in which specific serine residues were altered to alanine or aspartate. Phosphorylation patterns of these mutants were analyzed by two-dimensional gel electrophoresis, and their transcriptional activity was monitored by reporter assays. Essential phosphorylation events were mapped to amino acids 437–438 and a redundant set of sites at either amino acids 429–431 or 441. IRF7 recovered from infected cells was heterogeneously phosphorylated at these sites, and greater phosphorylation correlated with increased transactivation. Interestingly, a distinct serine cluster conserved in the related protein IRF3 was also essential for IRF7 activation and distal phosphorylation. However, the essential role of this motif did not appear to be fulfilled by phosphorylation. Rather, these serine residues and an adjacent leucine were required for phosphorylation at distal sites and may determine a conformational element required for function. Production of type I interferon in response to a wide variety of bacterial and viral infections is a major component of innate immunity and is essential to host defense against microbial invasion. Type I interferon (IFN) 1The abbreviations used are: IFN, interferon; IRF, interferon regulatory factor; ISG, interferon-stimulated gene; WT, wild type; NDV, Newcastle disease virus; TBK, TANK-binding kinase; IKK, IκB kinase. 1The abbreviations used are: IFN, interferon; IRF, interferon regulatory factor; ISG, interferon-stimulated gene; WT, wild type; NDV, Newcastle disease virus; TBK, TANK-binding kinase; IKK, IκB kinase. family consists of a single IFNβ gene and multiple IFNα genes that are clustered on mouse chromosome 4 (1Kelley K.A. Pitha P.M. Nucleic Acids Res. 1985; 13: 805-823Crossref PubMed Scopus (69) Google Scholar) and transcriptionally activated in virus-infected cells. Transcriptional induction of the IFNα/β genes is a rapid biphasic process in most cell types; initial induction of IFNα4 and -β is dependent on the constitutively expressed transcription factor IRF3 and subsequent induction of the other members of the IFNα family (IRF7-dependent) is achieved after positive feedback resulting in IRF7 protein production and activation (2Marié I. Durbin J.E. Levy D.E. EMBO J. 1998; 17: 6660-6669Crossref PubMed Google Scholar, 3Sato M. Hata N. Asagiri M. Nakaya T. Taniguchi T. Tanaka N. FEBS Lett. 1998; 441: 106-110Crossref PubMed Scopus (455) Google Scholar). Similar to many signaling pathways, the activating switch for transcription is achieved by phosphorylation of specific transcription factors. Transcription of the IFNβ gene is controlled by an enhanceosome composed of at least the AP1 transcription factor (c-jun-ATF2), phosphorylated by the c-Jun kinase, NFκB, released from its inhibitor IκB after phosphorylation-induced degradation through the action of the IκB kinase (composed of IKKα, IKKβ, and IKKγ/NEMO), and IRF3 and IRF7, activated by phosphorylation. The kinases thought responsible for phosphorylating IRF3 and IRF7 have been recently identified as distant members of the IKK family, TBK1 and IKK-ϵ (or IKKi) (4Fitzgerald K.A. McWhirter S.M. Faia K.L. Rowe D.C. Latz E. Golenbock D.T. Coyle A.J. Liao S.M. Maniatis T. Nat. Immunol. 2003; 4: 491-496Crossref PubMed Scopus (2003) Google Scholar, 5Sharma S. tenOever B.R. Grandvaux N. Zhou G.P. Lin R. Hiscott J. Science. 2003; 300: 1148-1151Crossref PubMed Scopus (1324) Google Scholar). Similarly to IRF3 and IRF7, TBK1 is constitutively expressed, whereas IKK-ϵ is mainly induced by stress stimuli, such as tumor necrosis factor α and lipopolysaccharide (6Pomerantz J.L. Baltimore D. EMBO J. 1999; 18: 6694-6704Crossref PubMed Google Scholar, 7Peters R.T. Liao S.M. Maniatis T. Mol. Cell. 2000; 5: 513-522Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar, 8Shimada T. Kawai T. Takeda K. Matsumoto M. Inoue J. Tatsumi Y. Kanamaru A. Akira S. Int. Immunol. 1999; 11: 1357-1362Crossref PubMed Scopus (305) Google Scholar). Phosphorylation takes place on a stretch of serine/threonine residues in the carboxyl-terminal regulatory domain of the proteins. The primary structure of these two proteins is closely related, and, notably, an SSL motif, which has been shown to be essential for transcriptional activity, is conserved between IRF3 and IRF7 of every studied species. However, the IRF7 regulatory domain contains many more serine/threonine residues, and the exact phosphorylation sites have yet to be determined precisely. Although considerable work has been reported concerning the target sites of IRF3, much less has been done regarding the identification of IRF7 phosphorylation sites. As it has been described for IRF3, our previous observation showed that mutation of the proximal set of serine residues 425 and 426 of murine IRF7 into alanine abrogated virus-induced phosphorylation and transcriptional activity (2Marié I. Durbin J.E. Levy D.E. EMBO J. 1998; 17: 6660-6669Crossref PubMed Google Scholar). However, we could not exclude the possibility that these residues were indispensable for proper conformation of the regulatory domain and that other residues were the bona fide targets of the virally induced kinases. Most recent work by Lin et al. (9Lin R. Mamane Y. Hiscott J. J. Biol. Chem. 2000; 275: 34320-34327Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar) and Yang et al. (10Yang H. Lin C.H. Ma G. Baffi M.O. Wathelet M.G. J. Biol. Chem. 2003; 278: 15495-15504Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar) on human IRF7 suggested that distal serine residues downstream of serines 425 and 426 could also play a role in virus-induced activation. However, Ser479, one residue believed to be a major target, is not conserved in murine IRF7 or IRF3. In this study, we investigated the relative importance of the proximal and distal sets of serine residues in the carboxyl-terminal regulatory domain of IRF7. We show that multiple serine residues are target for phosphorylation. In particular, two distinct groups of serine, Ser425 and Ser426 and Ser437 and Ser438, are indispensable for virally induced activation of IRF7 in a partially redundant manner. Furthermore, we demonstrate that secondary phosphorylation sites synergize with the primary targets, Ser437 and Ser438, to achieve maximal activation in response to viral infection. Moreover, we provide several lines of evidence that the essential nature of the proximal group of serine residues may not be mediated by phosphorylation. Instead, these residues are crucial for phosphorylation at distal sites possibly by maintaining the proper conformation of the carboxyl-terminal transactivation domain or by serving as a recognition motif for the virally activated kinases. 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 Newcastle disease was in (a kind gift from A. and viral infections were performed as described (2Marié I. Durbin J.E. Levy D.E. EMBO J. 1998; 17: 6660-6669Crossref PubMed Google Scholar). and murine IRF7 into and have been described (2Marié I. Durbin J.E. Levy D.E. EMBO J. 1998; 17: 6660-6669Crossref PubMed Google Scholar). and were kind from T. Maniatis R.T. Liao S.M. Maniatis T. Mol. Cell. 2000; 5: 513-522Abstract Full Text Full Text PDF PubMed Scopus (272) Google and the TBK1 was a kind gift from D. Baltimore (6Pomerantz J.L. Baltimore D. EMBO J. 1999; 18: 6694-6704Crossref PubMed Google Scholar). The IRF7 mutants were by by using and were by DNA In kinase for were performed after of the protein in 293T cells. using the recovered protein was in of kinase in the of of and of or mutant at a of or at for and analyzed by 10% and using a were at the using standard for were as described J. Levy D.E. PubMed Scopus Google Scholar). were gel and and at for for and by for were in and for at to on for the were to and by using of 293T cells were as described J. Levy D.E. PubMed Scopus Google Scholar). The was performed by nuclear of with a to the response element from the gene D.E. 1998; PubMed Scopus Google as described J. Levy D.E. Science. PubMed Scopus Google Scholar). IRF7 on induced phosphorylation is a to the conformational responsible for the transactivation of IRF7. work has mapped the phosphorylation events to a domain in the of IRF7 and shown that a mutant the amino acids was to viral (2Marié I. Durbin J.E. Levy D.E. EMBO J. 1998; 17: 6660-6669Crossref PubMed Google Scholar, 3Sato M. Hata N. Asagiri M. Nakaya T. Taniguchi T. Tanaka N. FEBS Lett. 1998; 441: 106-110Crossref PubMed Scopus (455) Google Scholar). phosphorylation was by analysis and of of IRF7. to an in IRF7 and the of a single and D. E. The regulatory domain of IRF7 contains serine residues that are potential phosphorylation sites However, which of these residues phosphorylated has not been of the used to IRF7 phosphorylation is the of a on Although to in this multiple potential sites of phosphorylation. to of the protein is two-dimensional gel As shown in IRF7 from cells be into three distinct Although these are to with did not their that phosphorylation is not the of the However, a analysis was performed on from cells, were of IRF7 were to phosphorylated after into the that IRF7 be phosphorylated at multiple distinct sites. also that not achieve phosphorylation of the to of several of phosphorylation. Interestingly, of either TBK1 or kinases responsible for IRF7 to shown by of the showed that of the three could be phosphorylated and that action of either kinase was to the phosphorylation of kinases did not to of the two-dimensional not of for Phosphorylation of determine the major targets of virally induced we group of serine residues in the regulatory domain into a residue that be The of phosphorylation of the mutants was monitored by two-dimensional gel analysis and after viral infection. analysis that of two distinct sets of serine residues, Ser425 and Ser426 and Ser437 and Ser438, IRF7 to viral infection. In mutation of the other two sets of and did not show a in the phosphorylation the of phosphorylation to be We that leucine residues are with these serine residues, a potential phosphorylation group of serine residues is either or by a leucine the importance of these leucine residues, we and into As shown in mutation of abrogated virally induced whereas mutant showed the phosphorylation as IRF7. this is with was the adjacent serine sets were in other it that the motif SSL acids has a role in virally induced whereas the motif acids has a Ser437 and the of determine the of group of we of The sets of serine residues were altered to a residue that the of a phosphorylated serine and for its function. by two-dimensional gel that these mutants an altered In we that most mutants were phosphorylated in response to viral infection, as by the of the of the Interestingly, the of mutant was not modified after viral that either this mutation the protein to viral or that these two serines were the targets for phosphorylation. is that Ser425 and Ser426 are the sites of phosphorylation the multiple after phosphorylation of the wild type protein of more two phosphorylated Moreover, mutant the serines Ser425 and Ser426 were into showed a the the phosphorylated protein and an analyzed from that these is that the distal serine residues are major targets for virus-induced whereas Ser425 and Ser426 are for kinase are either not phosphorylated or their is not by Transcriptional of IRF7 two-dimensional gel patterns were not to an concerning the of the residues, the of this study, it was essential to determine the of activity of mutant in and infected cells. this we cotransfected IRF7 and the mutants in COS cells with a reporter construct by the a target gene of IRF7 (2Marié I. Durbin J.E. Levy D.E. EMBO J. 1998; 17: 6660-6669Crossref PubMed Google Scholar). As shown IRF7 transactivation was viral (2Marié I. Durbin J.E. Levy D.E. EMBO J. 1998; 17: 6660-6669Crossref PubMed Google Scholar). However, mutation of Ser425 and Ser426 or Ser437 and abrogated virally induced activity Moreover, mutant also showed a of viral The other or were were not in their viral with are with was with the phosphorylation that virally induced phosphorylation is the key regulatory to transactivation ability. more was the activity of the mutants was with the that mutation of to did not have a on the virus-induced or transactivation mutation of the residues into either or and did not the mutants constitutively However, a of activity in the of to viral that these residues are or at most phosphorylated in response to viral infection. However, were to as by the could with the residues that are primary targets for the virus-activated kinases, transactivation as by the after mutation into showed a activity in of the with these residues the major targets for virally induced phosphorylation. Interestingly, mutation of serine residues 429–431 the mutant constitutively of activity in the of the wild type protein viral infection. Furthermore, we a mutant was into in to the in we a protein that a greater activity, IRF7 activated by viral infection. is to that activity of and was not increased by viral infection, that phosphorylation could We that phosphorylation of Ser437 and is required for virus-induced whereas phosphorylation of transactivation the of distal phosphorylation. the of the role of the set of Ser425 and that were to be essential to virally induced phosphorylation and activity I. Durbin J.E. Levy D.E. EMBO J. 1998; 17: 6660-6669Crossref PubMed Google and and Ser425 and Ser426 were into the resulting mutant did not activity to viral infection. is with our previous that this mutant could not be phosphorylated in response to viral and that the essential role of these serine is the of IRF7 of or increased activity of the mutants could be of of other that are a to IRF7 transactivation in the or DNA We tested the DNA of several As described I. E. A. Levy D.E. Mol. Cell. Biol. 2000; PubMed Scopus Google DNA by IRF7 was increased in Interestingly, constitutively mutants and showed increased DNA in with the DNA of was after viral infection, the of DNA in and are of was we monitored IRF7 activity by a between of DNA and transcriptional Essential of Ser425 and that the mutation of Ser425 and Ser426 to did not increased activity suggested that these residues not be targets of an essential role in IRF7 function. We their role proper conformation of the regulatory domain or as a recognition for the virally activated kinases, IKK-ϵ and this we the proximal group of Ser425 and Ser426 either to alanine or in the of the constitutively mutants and We the of the proximal set of serines was to provide a for the kinases that the distal group of serine and their mutation in the of constitutively mutants have a as shown in these partially the activity of the constitutively mutants mutant with and mutants and with that Ser425 and Ser426 were other serine residues in the regulatory domain for phosphorylation were into either that these serines are phosphorylation targets or that are for of the carboxyl-terminal transactivation determine these residues were phosphorylated in the of we the two-dimensional of with Ser425 and Ser426 are into As shown in the more in that may be partially phosphorylated on residues Ser425 and However, mutation of Ser425 and Ser426 into either or did not the activity of mutant and and a transactivation IRF7 in cells, of activation to that of viral activated IRF7 that Ser425 and Ser426 are to achieve maximal activity, these residues are not to a constitutively this did not exclude the possibility that these residues also as a recognition motif in the of the this we a the the proximal SSL motif, amino acids to the protein domain of the human type protein D. H. A. E. K. M. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). As a we also a mutant the that the SSL motif was into alanine The potential between the and the IRF7 kinase, was tested by the of IKK-ϵ in an in kinase in or of the or mutant IKK-ϵ in a that the kinase with the The mutant also showed of this did not show a that it was to be or et al. M. M. T. K. T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) recently that activity of to mutant in this was of the phosphorylation of the proximal set of serine residues and in the possibility of a in IRF7, we IRF7 and transactivation in the reporter using cells that could be of the kinase activity responsible for phosphorylating IRF7, TBK1 and this we used TBK1-deficient fibroblasts from and with a of (6Pomerantz J.L. Baltimore D. EMBO J. 1999; 18: 6694-6704Crossref PubMed Google to its In these cells of the kinase activity of IRF7 carboxyl-terminal regulatory IRF7 its activity, whereas activity As a TBK1-deficient cells with TBK1 showed a activity of IRF7, in the of kinases. In activity was greater in cells with suggested that activity was not of phosphorylation of Ser425 and Ser426 by TBK1 or whereas the activity of IRF7 is of of IKK In to determine Ser425 and Ser426 were targets of we a from the mutant As is constitutively activated by Moreover, our that and were not essential residues to achieve phosphorylation and and mutant the of and Ser425 and Ser426 as potential phosphorylation targets in response to viral infection. We tested the of such a mutant to be activated in response to viral infection. As shown in and mutant was not to infection, either by increased transcriptional activity or by altered Interestingly, as a major a more that was Ser425 and Ser426 were to in a of phosphorylation is with the that Ser425 and Ser426 are not primary targets for virally induced may phosphorylated and to the activity of IRF7. Furthermore, the groups of and the mutation of sets IRF7 that these serine residues are redundant with other that the of at least one or is crucial for viral of IRF7. induced phosphorylation is the crucial that the transactivation of IRF7. of the of IRF7 is that it contains potential sites clustered in its carboxyl-terminal regulatory have been concerning which residues are essential for virally dependent IRF7 activity and this that these residues are phosphorylated in (9Lin R. Mamane Y. Hiscott J. J. Biol. Chem. 2000; 275: 34320-34327Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar, H. Lin C.H. Ma G. Baffi M.O. Wathelet M.G. J. Biol. Chem. 2003; 278: 15495-15504Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). In this study, we provide evidence that Ser437 and are the primary targets for phosphorylation that phosphorylation on either residues of the group of or on is required to IRF7 in virus-infected cells. Furthermore, our that the proximal group of Ser425 and Ser426 is essential for not as phosphorylation targets also for a which phosphorylation at the major distal sites. may be as a recognition motif for the virally activated kinases or as a for proper conformation of IRF7 regulatory for a role for Ser425 and Ser426 is by several lines of The mutant is not constitutively The mutant that of the serines in is constitutively and its activity is not by viral infection. The mutant a activity in the of IRF7 kinases, that its activity is not of phosphorylation of Ser425 and whereas the activity of the protein is dependent on these kinases. The mutant the targets for phosphorylation are Ser425 and be activated in response to viral infection. our a in which phosphorylation of Ser425 and Ser426 is for transcriptional activity, the major phosphorylation targets, Ser437 and However, it not out the possibility that Ser425 and Ser426 phosphorylated to the phosphorylation of Ser437 and and to the phosphorylation and the transcriptional of activated IRF7. these serine residues with are required for the phosphorylation of the distal serine residues, possibly serving as a recognition motif for TBK1 and to a subsequent conformational the distal group of serines and for phosphorylation of the primary targets Ser437 and by phosphorylation of secondary targets and and possibly also Ser425 and Interestingly, the serine residues to Ser425 and Ser426 in IRF3 at an at the as in the recently IRF3 that these residues be to with other proteins K. M. M. Y. Y. H. Akira S. T. Nat. Biol. 2003; PubMed Scopus Google Scholar, H. R. R. Lin K. Nat. Biol. 2003; PubMed Scopus Google Scholar). are of has been reported for IRF3, with The major target of phosphorylation identified in this is the distal group of Ser437 and of these residues, Ser438, is conserved between IRF3 and IRF7 in the mouse and this residue to in human IRF3, which has been shown to play a mutant with protein after viral and the with protein and is a of IRF3 target genes Grandvaux N. tenOever B.R. D. Lin R. Hiscott J. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). which has been identified as the major target for in phosphorylation of IRF3, is not in IRF7 Grandvaux N. tenOever B.R. D. Lin R. Hiscott J. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). However, these have been by et al. M. M. T. K. T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google a in which the proximal group is the target of the IRF3 kinases. In either IRF3 and IRF7 appear to be phosphorylated on of the of IRF7 with IRF3 is its activity in of viral infection. We show that this activity is of activation of the kinases the possibly through a IRF7 not activity cotransfected with in TBK1-deficient cells Interestingly, the the activity of the mutant is a activation of the observation that activity is not to phosphorylation of the proximal group of serine, Ser425 and phosphorylation of this set of serines is achieved by an kinase distinct from TBK1 and However, phosphorylation of Ser425 and Ser426 by a yet to be identified kinase is the for IRF7 as by et al. M. M. T. K. T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) for IRF3, the in IFNα induction in TBK1-deficient cells be to H. S. M. T. H. Kawai T. K. Takeda K. Akira S. J. PubMed Scopus Google Scholar, G. J. PubMed Scopus Google Scholar, S.M. K.A. J. Rowe D.C. Golenbock D.T. Maniatis T. S. A. PubMed Scopus Google Scholar, B.R. S. Grandvaux N. I. H. M. Akira S. Lin R. Hiscott J. J. PubMed Scopus Google Scholar). Interestingly, an for IRF7 action was recently in which its transcriptional activity is by with and in response to T. S. H. M. K. M. Inoue J. S. Akira S. Nat. Immunol. 5: PubMed Scopus Google Scholar, K. H. T. H. N. N. Y. A. Taniguchi T. S. A. PubMed Scopus Google Scholar). In this kinases not to be and a role for carboxyl-terminal phosphorylation was not established. Moreover, it is that proteins are in the response of IRF7 to viral D.E. Nat. Immunol. 5: PubMed Scopus Google Scholar). In this study, analysis of by two-dimensional gel was performed for the We have been to determine that several of phosphorylation after viral in the The by the mutants that IRF7 transcriptional activity is to the of phosphorylation of the with the transcriptional activity of IRF7 resulting from the of the phosphorylation from our is that the of mediated by IRF7 may be dependent on the of phosphorylation. We that the activity of IRF7 be as a of its of phosphorylation as a of the host on the of an viral as by the and of kinase We W.-C. A. T. and D. Baltimore for the gift of cell and We for for and and for