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J. Biol. Chem., Vol. 282, Issue 31, 22816-22822, August 3, 2007
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12
From the
Department of Biological Chemistry and Molecular Pharmacology, Howard Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts 02115 and Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138
Received for publication, April 10, 2007 , and in revised form, May 23, 2007.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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and/or TBK1. Phosphorylation results in IRF-3 dimerization and removal of an autoinhibitory structure to allow interaction with the coactivators CBP/p300. The precise role of the different phosphorylation sites has remained controversial. Using purified proteins we show that TBK1 can directly phosphorylate full-length IRF-3 in vitro. Phosphorylation at residues in site 2 (Ser396—Ser405) alleviates autoinhibition to allow interaction with CBP (CREB-binding protein) and facilitates phosphorylation at site 1 (Ser385 or Ser386). Phosphorylation at site 1 is, in turn, required for IRF-3 dimerization. The data support a two-step phosphorylation model for IRF-3 activation mediated by TBK1. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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and 
(IFN- and IFN-), the chemokine RAN-TES (regulated on activation normal T cell expressed and secreted), and many other genes that respond to viral and bacterial infection (1-4). In the absence of infection, IRF-3 is in a latent conformation in the cytoplasm (1, 5). Upon infection, the host detects an invading pathogen through pattern recognition receptors such as the toll-like receptors or the cytoplasmic receptors RIG-I and Mda-5. Depending on the microbial pathogen, distinct signaling pathways are triggered, many of which lead to activation of IRF-3. IRF-3 is activated by phosphorylation mediated directly or as part of a signaling cascade by the I
B kinase (IKK)-related kinases, TBK1 and IKK (6-10). Phosphorylation of IRF-3 leads to dimerization, translocation to the nucleus, and association with the coactivator CREB-binding protein (CBP) or the closely related p300 (1, 11-13).
IRF-3 shares a conserved N-terminal DNA-binding domain with other members of the interferon response factor family of transcription factors (14). It also contains a C-terminal IRF association domain (IAD), which mediates phosphorylation-dependent homo- or hetero-oligomerization with other IRF family members and interaction with CBP/p300 (15). The crystal structure of the N-terminal DNA-binding domain bound to the interferon- promoter and the crystal structure of the IAD have been determined (14-16). Activation of IRF-3 requires phosphorylation on a cluster of serine and threonine residues in the C-terminal region of the IAD spanning amino acids 385 to 405. Despite considerable efforts, using mutagenesis, phosphopeptide mapping, and phospho-specific antibodies, the exact position and role of phosphorylation sites in the 385-405 region remain controversial. On the one hand, Fujita and co-workers (17) suggest that phosphorylation at Ser385/Ser386 (site 1) is key to the activation of IRF-3 and that phosphorylation at site 2, spanning residues 396 to 405, has an auxiliary but not essential role. This view is supported by the observation that mutation of Ser385 or Ser386 to Ala abolishes IRF-3 activity (5, 12). In addition, Fujita and co-workers (16, 17) used a phosphospecific antibody against Ser386 to show that overexpressed IRF-3, activated by virus or by overexpression of upstream regulators, is phosphorylated on Ser386 and that phosphorylation on Ser386 is observed in the dimeric form of IRF-3. On the other hand, Hiscott and colleagues (11) argue that phosphorylation of site 2 residues may also be critical for virus-inducible IRF-3 activation. For instance, substitutions of the site 2 residues Ser396, Ser398, Ser402, Thr404, and Ser405 with the phosphomimetic Asp or Glu (IRF-3 5E) lead to a constitutive phenotype (11). Further mutagenesis and use of a phosphospecific antibody has identified Ser396 as a key site 2 residue that is phosphorylated in vivo upon virus infection (18). Substitution of Ser385 and Ser386 with either Ala or Asp inhibits IRF-3 activation (5). Taken together, these data point to a phosphorylation-dependent mechanism of IRF-3 activation that requires serine 386 and a cluster of serine/threonine residues between Ser396 and Ser405.
Analysis of the crystal structure of the IAD by Qin et al. (15) suggested that phosphorylation of the site 2 residues 396-405 results in unfolding of an N-and C-terminal autoinhibitory structure, essentially consistent with the functional studies of Hiscott and colleagues (5, 18). Unfolding of the autoinhibitory structure by introduction of a negative charge, through either phosphorylation or mutagenesis, exposes a hydrophobic surface for interaction with CBP/p300. Qin et al. (15) also suggested that the dimer in the asymmetric unit of the crystal was not physiologically relevant and that the phosphorylated, rearranged C-terminal tail might be involved in subunit dimerization. In contrast, Takahasi et al. (16) doubt the existence of the N- and C-terminal autoinhibitory structure and interpret their crystallographic results to be consistent with the functional studies of Fujita and co-workers (12). They suggest that the dimer in the asymmetric unit is physiologically relevant and that phosphorylation of Ser385/Ser386 results directly in IAD dimerization. Dimerization would generate a combined surface for recognition of CBP/p300 (16). This latter model was recently ruled out by a co-complex crystal structure of the IAD with the interferon-binding domain (IBiD) of CBP (19). This structure shows that removal of the autoinhibitory N and C termini of the IAD leads to exposure of a binding pocket that interacts with IBiD and that the IAD and IBiD form a 1:1 complex.
To clarify the role of phosphorylation in IRF-3 activation, as well as the role of TBK1 in this process, we purified to homogeneity full-length IRF-3, the constitutive mutant IRF-3 5E, and the kinase TBK1. We found that, as predicted, purified IRF-3 is a monomer in solution and that IRF-3 5E is a dimer. Access to highly purified and homogeneous protein preparations allowed us to map the phosphorylation sites in more detail. Monomeric IRF-3 is phosphorylated neither at site 1 nor at site 2. Dimeric IRF-3 5E is fully phosphorylated at site 1 (Ser385 or Ser386) as a result of constitutive in vivo phosphorylation by an insect cell kinase. TBK1 can directly phosphorylate IRF-3, resulting in a distribution of monomeric and dimeric IRF-3, consistent with earlier findings (5, 20). Mapping of the phosphorylation sites in a purified TBK1-phosphorylated IRF-3 dimer showed 100% phosphorylation of one serine residue at site 1 (Ser385 or Ser386) and of one serine/threonine residue at site 2 (Thr404 or Ser405). The purified TBK1-phosphorylated IRF-3 monomer is not phosphorylated at site 1; it is fully phosphorylated on one serine at site 2 (Thr404 or Ser405), but mutation of residues 404 and 405 to Ala does not completely abolish in vitro phosphorylation of site 2 residues. We also show that CBP binding requires phosphorylation at site 2, the residues of which are functionally redundant. Thus, modification in site 2 is critical for alleviating autoinhibition, for allowing interaction with CBP, and for facilitating phosphorylation at site 1. Phosphorylation at site 1 leads in turn to IRF-3 dimerization as proposed previously (17).
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Purification of Full-length IRF-3 and IRF-3 5E from Insect Cells—Recombinant baculovirus-infected cell pellets were lysed by freezing and then thawed in equilibration buffer containing 20 mM HEPES (pH 7.5), 500 mM NaCl, 1 mM EDTA, 1mM phenylmethylsulfonyl fluoride, and 0.5% Nonidet P-40. For TBK1 purification, frozen cell pellets were thawed and resuspended in 20 mM HEPES (pH 7.5), 500 mM NaCl, 0.5 mM sodium fluoride, 1 mM EDTA, 0.1% Tween 20, and Complete protease inhibitors (Roche Applied Science). The cells were homogenized by sonication and the lysate clarified by centrifugation at 180,000 x g for 40 min. The clarified insect cell lysate was incubated with pre-equilibrated Anti-FLAG M2-agarose affinity gel (Sigma) for 1 h and then applied to a 25-ml Econo column (Bio-Rad). The resin was washed with 200 ml of equilibration buffer, and then bound protein was eluted using 0.1 mg/ml triple FLAG peptide (sequence, MDYKDHDADYKDHDIDYKDDDDK) dissolved in equilibration buffer.
Fractions were concentrated using a Centricon-plus 30 ultrafiltration unit (Amicon, Inc.) and further purified on a Hi-Load 16/60 Superdex 200 gel filtration column (GE Healthcare) equilibrated in 20 mM HEPES (pH 7.5), 150 mM NaCl, 1 mM dithiothreitol (DTT). The final protein was concentrated to 40 mg/ml for IRF-3, 14 mg/ml for IRF-3 5E, and 2.5 mg/ml for TBK1 in a Centricon-plus 30 ultrafiltration unit; aliquots were flash-frozen in liquid N2 and stored at -80 °C. Protein concentrations were determined by diluting the sample in 10 mM KH2PO4 (pH 6.8), 6 M guanidine HCL and measuring the absorbance at 280 nm using a molar extinction coefficient calculated from the amino acid sequence.
IRF-3 Phosphorylation with TBK1—600 pmol of IRF-3 was incubated for 30 min at 30 °C with 24 pmol of TBK1 in a buffer consisting of 20 mM HEPES (pH 7.6), 50 mM NaCl, 20 mM -glycerol phosphate, 1 mM sodium vanadate, 10 mM MgCl2, 1 mM DTT, and 1 mM ATP.
Analytical Gel Filtration—IRF-3, IRF-3 5E, and IRF-3 phosphorylated by TBK1 were further analyzed, on a Superdex 200 10/300 gel filtration column (GE Healthcare) equilibrated in 20 mM HEPES (pH 7.5), 150 mM NaCl, and 1 mM DTT. The column was calibrated using a set of globular protein standards consisting of thyroglobulin (670 kDa), 
-globulin (158 kDa), ovalbumin (44 kDa), and myoglobin (17 kDa) (Bio-Rad). Calibration curves were constructed by plotting Kav versus ln (molecular weight), where Kav equals (Ve - V0)/(Vt - V0). Ve is the measured elution volume of the markers. V0, the void volume of the column, was determined by gel filtration of blue dextran, and Vt, the total column volume, was specified by the manufacturer (GE Healthcare).
Tryptic Digest—Stocks of trypsin were made from a lyophilized powder (Sigma T-8253) to 10 mg/ml in 1 mM HCl, 20 mM CaCl2, and aliquots were flash-frozen in liquid N2 and stored at -80 °C. IRF-3 and IRF-3 5E were diluted to 1.25 mg/ml in 80 µl of 25 mM Tris-HCl (pH 8.5), 150 mM NaCl, and 1 mM DTT. Trypsin was diluted to 0.5 mg/ml before use, and the digest was started by adding 1 µl of protease (ratio of protease:substrate was 1:200) to the mix. At each time point trypsin was inactivated with phenylmethylsulfonyl fluoride and EDTA and boiling of the sample for 5 min in SDS loading buffer. The samples were analyzed on a 4-20% gradient SDS-PAGE. For Edman sequencing, the protein was transferred onto polyvinylidene difluoride membrane. Coomassie-stained bands were excised and sequenced at the Tufts University Core Facility (Boston).
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Assessment of Phosphorylation—10-µg aliquots of each protein were desalted by microbore reverse phase liquid chromatography on a 300A polystyrene-divinylbenzene column. Intact protein phosphorylation load was estimated by ESI ion trap (Bruker-Agilent) and by MALDI-TOF mass spectrometry prior to tryptic digestion aided by microwave irradiation (CEM Corp.; 5 min, 50 °C, 25 watts). Individual peptide phosphorylation levels were measured simultaneously by flow injection of the entire desalted tryptic digest and analysis of ESI-FTICR spectra (Bruker 9.4T). The accuracy of this method was confirmed by spiking the tryptic digest with known amounts of two pairs of mono- and diphosphorylated versus nonphosphorylated peptides. Phosphorylation sites were deduced by collision-induced dissociation time-of-flight tandem mass spectroscopy (CID TOF MS/MS; AB 4700).
Purification of IBiD and in Vitro Protein Interaction Assay—IBiD was overexpressed in Escherichia coli BL21(DE3) with an N-terminal His tag. The protein was purified using nickel-nitrilotriacetic acid resin (Qiagen) followed by reversed-phase high pressure liquid chromatography and verified by electrospray ionization mass spectrometry. 
50 mg of purified IBiD was bound to 1 ml of 50% nickel-nitrilotriacetic acid resin in 20 mM Tris (pH 8.0), 300 mM NaCl, 20 mM imidazole, and excess protein was removed by washing in the same buffer. For each binding reaction 10 µg of each of the different IRF-3 preparations was incubated with 20 µl of IBiD-charged nickel-nitrilotriacetic acid resin in a total volume of 40 µl. The reaction was incubated at 4 °C for 30 min, and a 10-µl sample (input) was removed. The beads were collected by a 1-min centrifugation at 500 x g, and a 10-µl sample of the supernatant was taken. The beads were than washed twice with 500 µl of 20 mM Tris (pH 8.0), 300 mM NaCl, 30 mM imidazole, and finally bound protein was eluted by boiling in SDS-loading buffer. The protein samples were resolved on a 4-20% gradient SDS-PAGE.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Analytical Ultracentrifugation of IRF-3 and IRF-3 5E—We performed sedimentation equilibrium experiments on IRF-3 and IRF-3 5E to determine the mass and oligomerization state independently of shape and matrix interactions. Both samples were analyzed at three different starting concentrations and three different rotor speeds. For all nine data sets, absorbance data were monitored at 280 nm and the resulting curves fitted using a nonlinear least-squares algorithm (Fig. 2). For both IRF-3 and IRF-3 5E, the residuals showed random distribution, indicating that the samples contained a single monodisperse species. For IRF-3, the data yielded a molecular mass of 52 ± 2 kDa, close to the calculated molecular mass of 48.2 kDa, showing that IRF-3 is a monomer. For IRF-3 5E, we obtained a molecular mass of 103 ± 2 kDa, about double the mass calculated from the composition. We conclude that IRF-3 5E is a dimer.
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Interaction with CBP—To determine the effect of IRF-3 phosphorylation on the interaction with CBP, we performed pulldown experiments using immobilized IBiD from CBP as bait. As shown in Fig. 4, TBK1-phosphorylated IRF-3 monomer (phosphorylated on site 2) and dimer (phosphorylated on site 1 and site 2) and the constitutive dimer IRF-3 5E (phosphorylated on site 1) all interacted with IBiD, whereas the full-length unphosphorylated IRF-3 or the N-terminal DNA-binding domain of IRF-3 did not. That is, introduction of a negative charge at site 2, either through phosphomimetic mutations (IRF-3 5E) or through TBK1-mediated phosphorylation, is sufficient to alleviate autoinhibition and allow interaction with IBiD. Dimerization, induced by phosphorylation of site 1, was not required for IBiD binding.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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TBK1 fully phosphorylated site 2, whereas phosphorylation at site 1 ranged between 20 and 50%, as measured by IRF-3 dimerization. We suggest that in vitro, TBK1 phosphorylates IRF-3 by a two-step sequential mechanism, in which site 2 is phosphorylated first. Introduction of negative charge in the site 2 residues leads to structural rearrangement of the autoinhibitory segments, and subsequent phosphorylation of site 1 leads to dimerization (Fig. 5). Although our mass spectrometry data could not distinguish which of the two site 1 residues is phosphorylated, it is likely to be Ser386, based on previous work (17) and our unpublished findings.4
Although it is well established that TBK1 activates IRF-3 by direct phosphorylation of the C-terminal residues (8, 21), it remains unclear whether TBK1 is the only kinase required for full IRF-3 phosphorylation at sites 1 and 2 in vivo. Whereas phosphorylation at site 2 is virus-inducible, several reports, as well as data presented here, indicate that site 1 can be constitutively phosphorylated in 293T cells or insect cells when phosphomimetic mutations are present in site 2 (16, 17). Therefore, we suggest that alleviation of autoinhibition by TBK1 phosphorylation of site 2 leads to a structure that is then efficiently phosphorylated at site 1 by TBK1 or by another kinase. The requirement of a second kinase would be consistent with the observation that TBK1 alone is not sufficient for full IRF-3 activation by Toll-like receptor 3 in vivo and that a second signaling pathway mediated by phophatidylinositol-3 kinase or by protein kinase C may be required (22, 23). We have no direct evidence for either of these pathways, however.
We show here that phosphorylation of site 2 by TBK1 results in monomeric IRF-3 that is competent to bind CBP. Thus, relief of autoinhibition, but not dimerization, is necessary and sufficient for interaction with CBP. This result is consistent with the crystal structure of IRF-3 in complex with the IBiD of CBP. Both the crystal structure and analytical ultracentrifugation reveal a 1:1 complex between IRF-3 and CBP (19). A 1:1 complex might not be physiologically relevant, because the predominant species of IRF-3 after virus infection is dimeric. A monomeric IRF-3·CBP complex could, however, interact with other IRFs such as IRF-3 or IRF-7 to form homo- or heterodimeric complexes, as suggested previously (1). Our results are consistent with the observation that a S385A/S386A mutant is dimerization-deficient (16). However, our data apparently contradict coimmunoprecipitation experiments showing that a S385A/S386A or a S386A mutant is unable to bind CBP in a virus-inducible manner (5, 18).4 These mutants should be dimerization-deficient but should be able to bind CBP after virus-induced phosphorylation of the site 2 residues. It is possible that an IRF-3 monomer binds weakly to CBP in vivo and that IRF-3 dimerization through phosphorylation of site 1 stabilizes this complex. We also observed that the phosphorylated IRF-3 dimer binds more strongly to DNA than does the phosphorylated IRF-3 monomer (data not shown), supporting the idea that dimeric IRF complexes are required for high affinity binding to CBP and to DNA in vivo.
There is inherent functional redundancy among the different site 2 residues, as demonstrated by the properties of phosphomimetic or alanine mutations. For example, mutation of Ser396 or Ser398 to Ala greatly reduces the virus inducibility of IRF-3, whereas introduction of phosphomimetic Asp at these residues is sufficient to generate a constitutive phenotype (11, 18). Similarly, substitution of Ser402, Thr404, or Ser405 with Ala reduces virus inducibility, whereas substitution with Asp generates a constitutive phenotype (11, 18). TBK1 phosphorylates an alternative site 2 residue when Thr404 and Ser405 have been changed to alanine, but our mass spectrometry data do not distinguish which of the three remaining site 2 residues is phosphorylated. The crystal structure of latent IRF-3 has shown that the site 2 residues are part of the autoinhibitory structure and that some of them are partially buried in a hydrophobic environment (15, 16). A plausible activation mechanism, consistent with the structures, is that phosphorylation enforces unfolding of the autoinhibitory elements and consequent exposure of helices 3 and 4 for interaction with the IBiD of CBP (15, 19).
In different cellular contexts, different residues in site 2 appear to be the major targets for activation. Redundancy at site 2 is probably important for a robust and context-independent response, as different signaling pathways will activate different spectra of protein kinases. The identity of the residue within site 2 that is the principal phosphorylation target depends on the specific inducer and cell line used in an experiment, as do the consequences of its modification for phosphorylation of site 1. For example, Ser396 is phosphorylated in U373/CD14 astrocytes in response to treatment with Sendai virus and poly(I-C) but not in response to treatment with lipopolysaccharide (18). The IRF 5E variant is active in human 293 cells but much less active in L929 cells (5, 20). These latter observations imply that constitutive phosphorylation of site 1 in the context of the 5E mutation is cell-line dependent, presumably because of different constitutive levels of the relevant kinase activities. We have found in the work described here that the principal residues phosphorylated by TBK1 in vitro are Thr404 and Ser405. These residues are in the turn between strand 13 and helix 5, part of the hydrophobic core of the autoinhibitory structure (15). Thr 404 forms an N-cap for helix 5 by hydrogen bonding to the backbone amide of Gln407; Ser405 has contacts with Lys409, Asp406, and Tyr408. Phosphorylation of either residue 404 or 405 is likely to produce rearrangement of the autoinhibitory structure and unmasking of the hydrophobic surfaces of helices 3 and 4, for interaction with the IBiD of CBP. In vitro kinase assays using a glutathione S-transferase-fused IRF-3 peptide, residues 380-427, indicate that Ser402 is the primary TBK1 target site, but the peptide probably resembles a partially or completely unfolded state of the autoinhibitory region, and it may not represent a principal intracellular conformation. In some cells, phosphorylation of Ser396 appears to be the most common modification. Substitution of Ser396 with Asp generates a constitutively active form of IRF-3, and mutation to Ala greatly reduces the virus inducibility of IRF-3-regulated genes (18). Serine396 is in the loop L6. It forms a hydrogen bond with the main-chain amide of Glu201 and potentially with the side-chain of Glu200. We therefore expect that phosphorylation of Ser396, like modification of Thr404 or Ser405, will destabilize the autotinhibitory structure (16). In short, the structure of the autoinhibitory region has shown that modification of any of the site 2 residues will trigger the unmasking of the IBiD site and potentiate phosphorylation of site 1; the region appears to have evolved in response to a variety of activating signals and a correspondingly varied spectrum of intracellular responses.
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FOOTNOTES |
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This article was selected as a Paper of the Week.
1 Present address: Dept. of Molecular and Cell Biology, University of California, Berkeley, 415 Life Sciences Addition, Berkeley, CA 94720.
2 To whom correspondence should be addressed. Tel.: 617-432-5607; Fax: 617-432-5600; E-mail: harrison{at}crystal.harvard.edu.
3 The abbreviations used are: IRF, interferon regulatory factor; IBiD, interferon-binding domain; CREB, cAMP-response element-binding protein; CBP, CREB-binding protein; IAD, IRF association domain; DTT, dithiothreitol; MOPS, 4-morpholinepropanesulfonic acid; TBK1, Tank-binding kinase 1.
4 S. M. McWhirter and T. Maniatis, unpublished observation.
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ACKNOWLEDGMENTS |
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