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• W2024592805 abstract "The transcription factor interferon regulatory factor 3 (IRF-3) regulates genes in the innate immune response. IRF-3 is activated through phosphorylation by the kinases IKKɛ 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. The transcription factor interferon regulatory factor 3 (IRF-3) regulates genes in the innate immune response. IRF-3 is activated through phosphorylation by the kinases IKKɛ 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. The transcription factor IRF-3 3The 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. is a central component of the innate immune system. It is required for expression of type I interferons α and β (IFN-α and IFN-β), the chemokine RAN-TES (regulated on activation normal T cell expressed and secreted), and many other genes that respond to viral and bacterial infection (1Wathelet M.G. Lin C.H. Parekh B.S. Ronco L.V. Howley P.M. Maniatis T. Mol. Cell. 1998; 1: 507-518Abstract Full Text Full Text PDF PubMed Scopus (654) Google Scholar, 2Hiscott J. Pitha P. Genin P. Nguyen H. Heylbroeck C. Mamane Y. Algarte M. Lin R. J. Interferon Cytokine Res. 1999; 19: 1-13Crossref PubMed Scopus (196) Google Scholar, 3Genin P. Algarte M. Roof P. Lin R. Hiscott J. J. Immunol. 2000; 164: 5352-5361Crossref PubMed Scopus (189) Google Scholar, 4Barnes B. Lubyova B. Pitha P.M. J. Interferon Cytokine Res. 2002; 22: 59-71Crossref PubMed Scopus (273) Google Scholar). In the absence of infection, IRF-3 is in a latent conformation in the cytoplasm (1Wathelet M.G. Lin C.H. Parekh B.S. Ronco L.V. Howley P.M. Maniatis T. Mol. Cell. 1998; 1: 507-518Abstract Full Text Full Text PDF PubMed Scopus (654) Google Scholar, 5Lin R. Mamane Y. Hiscott J. Mol. Cell. Biol. 1999; 19: 2465-2474Crossref PubMed Scopus (271) Google Scholar). 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 IκB kinase (IKK)-related kinases, TBK1 and IKKɛ (6Sharma S. tenOever B.R. Grandvaux N. Zhou G.P. Lin R. Hiscott J. Science. 2003; 300: 1148-1151Crossref PubMed Scopus (1366) Google Scholar, 7Fitzgerald 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 (2085) Google Scholar, 8McWhirter S.M. Fitzgerald K.A. Rosains J. Rowe D.C. Golenbock D.T. Maniatis T. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 233-238Crossref PubMed Scopus (451) Google Scholar, 9Hemmi H. Takeuchi O. Sato S. Yamamoto M. Kaisho T. Sanjo H. Kawai T. Hoshino K. Takeda K. Akira S. J. Exp. Med. 2004; 199: 1641-1650Crossref PubMed Scopus (464) Google Scholar, 10Perry A.K. Chow E.K. Goodnough J.B. Yeh W.C. Cheng G. J. Exp. Med. 2004; 199: 1651-1658Crossref PubMed Scopus (312) Google Scholar). 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 (1Wathelet M.G. Lin C.H. Parekh B.S. Ronco L.V. Howley P.M. Maniatis T. Mol. Cell. 1998; 1: 507-518Abstract Full Text Full Text PDF PubMed Scopus (654) Google Scholar, 11Lin R. Heylbroeck C. Pitha P.M. Hiscott J. Mol. Cell. Biol. 1998; 18: 2986-2996Crossref PubMed Scopus (756) Google Scholar, 12Yoneyama M. Suhara W. Fukuhara Y. Fukuda M. Nishida E. Fujita T. EMBO J. 1998; 17: 1087-1095Crossref PubMed Scopus (690) Google Scholar, 13Weaver B.K. Kumar K.P. Reich N.C. Mol. Cell. Biol. 1998; 18: 1359-1368Crossref PubMed Scopus (297) Google Scholar). IRF-3 shares a conserved N-terminal DNA-binding domain with other members of the interferon response factor family of transcription factors (14Panne D. Maniatis T. Harrison S.C. EMBO J. 2004; 23: 4384-4393Crossref PubMed Scopus (133) Google Scholar). 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 (15Qin B.Y. Liu C. Lam S.S. Srinath H. Delston R. Correia J.J. Derynck R. Lin K. Nat. Struct. Biol. 2003; 10: 913-921Crossref PubMed Scopus (174) Google Scholar). The crystal structure of the N-terminal DNA-binding domain bound to the interferon-β promoter and the crystal structure of the IAD have been determined (14Panne D. Maniatis T. Harrison S.C. EMBO J. 2004; 23: 4384-4393Crossref PubMed Scopus (133) Google Scholar, 15Qin B.Y. Liu C. Lam S.S. Srinath H. Delston R. Correia J.J. Derynck R. Lin K. Nat. Struct. Biol. 2003; 10: 913-921Crossref PubMed Scopus (174) Google Scholar, 16Takahasi K. Suzuki N.N. Horiuchi M. Mori M. Suhara W. Okabe Y. Fukuhara Y. Terasawa H. Akira S. Fujita T. Inagaki F. Nat. Struct. Biol. 2003; 10: 922-927Crossref PubMed Scopus (129) Google Scholar). 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 (17Mori M. Yoneyama M. Ito T. Takahashi K. Inagaki F. Fujita T. J. Biol. Chem. 2004; 279: 9698-9702Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar) 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 (5Lin R. Mamane Y. Hiscott J. Mol. Cell. Biol. 1999; 19: 2465-2474Crossref PubMed Scopus (271) Google Scholar, 12Yoneyama M. Suhara W. Fukuhara Y. Fukuda M. Nishida E. Fujita T. EMBO J. 1998; 17: 1087-1095Crossref PubMed Scopus (690) Google Scholar). In addition, Fujita and co-workers (16Takahasi K. Suzuki N.N. Horiuchi M. Mori M. Suhara W. Okabe Y. Fukuhara Y. Terasawa H. Akira S. Fujita T. Inagaki F. Nat. Struct. Biol. 2003; 10: 922-927Crossref PubMed Scopus (129) Google Scholar, 17Mori M. Yoneyama M. Ito T. Takahashi K. Inagaki F. Fujita T. J. Biol. Chem. 2004; 279: 9698-9702Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar) 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 (11Lin R. Heylbroeck C. Pitha P.M. Hiscott J. Mol. Cell. Biol. 1998; 18: 2986-2996Crossref PubMed Scopus (756) Google Scholar) 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 (11Lin R. Heylbroeck C. Pitha P.M. Hiscott J. Mol. Cell. Biol. 1998; 18: 2986-2996Crossref PubMed Scopus (756) Google Scholar). 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 (18Servant M.J. Grandvaux N. tenOever B.R. Duguay D. Lin R. Hiscott J. J. Biol. Chem. 2003; 278: 9441-9447Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). Substitution of Ser385 and Ser386 with either Ala or Asp inhibits IRF-3 activation (5Lin R. Mamane Y. Hiscott J. Mol. Cell. Biol. 1999; 19: 2465-2474Crossref PubMed Scopus (271) Google Scholar). 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. (15Qin B.Y. Liu C. Lam S.S. Srinath H. Delston R. Correia J.J. Derynck R. Lin K. Nat. Struct. Biol. 2003; 10: 913-921Crossref PubMed Scopus (174) Google Scholar) 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 (5Lin R. Mamane Y. Hiscott J. Mol. Cell. Biol. 1999; 19: 2465-2474Crossref PubMed Scopus (271) Google Scholar, 18Servant M.J. Grandvaux N. tenOever B.R. Duguay D. Lin R. Hiscott J. J. Biol. Chem. 2003; 278: 9441-9447Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). 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. (15Qin B.Y. Liu C. Lam S.S. Srinath H. Delston R. Correia J.J. Derynck R. Lin K. Nat. Struct. Biol. 2003; 10: 913-921Crossref PubMed Scopus (174) Google Scholar) 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. (16Takahasi K. Suzuki N.N. Horiuchi M. Mori M. Suhara W. Okabe Y. Fukuhara Y. Terasawa H. Akira S. Fujita T. Inagaki F. Nat. Struct. Biol. 2003; 10: 922-927Crossref PubMed Scopus (129) Google Scholar) 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 (12Yoneyama M. Suhara W. Fukuhara Y. Fukuda M. Nishida E. Fujita T. EMBO J. 1998; 17: 1087-1095Crossref PubMed Scopus (690) Google Scholar). 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 (16Takahasi K. Suzuki N.N. Horiuchi M. Mori M. Suhara W. Okabe Y. Fukuhara Y. Terasawa H. Akira S. Fujita T. Inagaki F. Nat. Struct. Biol. 2003; 10: 922-927Crossref PubMed Scopus (129) Google Scholar). This latter model was recently ruled out by a co-complex crystal structure of the IAD with the interferon-binding domain (IBiD) of CBP (19Qin B.Y. Liu C. Srinath H. Lam S.S. Correia J.J. Derynck R. Lin K. Structure (Camb.). 2005; 13: 1269-1277Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). 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 (5Lin R. Mamane Y. Hiscott J. Mol. Cell. Biol. 1999; 19: 2465-2474Crossref PubMed Scopus (271) Google Scholar, 20Suhara W. Yoneyama M. Iwamura T. Yoshimura S. Tamura K. Namiki H. Aimoto S. Fujita T. J. Biochem. (Tokyo). 2000; 128: 301-307Crossref PubMed Scopus (66) Google Scholar). 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 (17Mori M. Yoneyama M. Ito T. Takahashi K. Inagaki F. Fujita T. J. Biol. Chem. 2004; 279: 9698-9702Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). Insect Cell Culture and Baculovirus Cultivation—The recombinant baculoviruses were propagated in Sf21 insect cells with Hinks TMN-FH insect cell medium (JRH Biosciences) supplemented with 10% fetal bovine serum (JRH Biosciences). Virus titration, amplification, and storage were performed as described previously. For production of recombinant IRF-3 and IRF-3 5E, Sf21 insect cells were infected with recombinant baculovirus at a multiplicity of infection of 2.0, maintained in spinner flasks at 28 °C, and harvested by centrifugation at 64 h postinfection. For production of TBK1, Hi5 insect cells were infected with recombinant baculovirus and otherwise treated as described above. 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 × 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 β-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), γ-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). Analytical Ultracentrifugation—Samples for analytical ultracentrifugation were dialyzed against 10 mm MOPS (pH 7.5), 150 mm NaCl, and 0.25 mm Tris (2-carboxyethyl)phosphine hydrochloride (TCEP) using a 3500 molecular weight cutoff dialysis membrane (Pierce). The dialysis buffer was used as a blank and as diluent for centrifugation. Analytical centrifugation was performed using an Optima XL-A analytical ultracentrifuge (Beckman Coulter) with an AN-60 Ti rotor. Six-channel cells were used, and data were acquired at a resolution of 0.001 cm with five replicates at a temperature of 4 °C. Absorbance at 280 nm was used to monitor concentration gradients. IRF-3 concentrations were 9, 4.5, and 2 μm, and the speeds were 12,000, 15,000, and 22000 rpm. IRF-3 5E concentrations were 8, 5, and 2 μm, and the concentration distribution was measured at three different rotor speeds, 8,500, 12,000, and 15,000 rpm. Samples were determined to have reached equilibrium when scans taken 4 h apart showed no systematic differences. The protein partial specific volume was calculated from the amino acid composition to 0.7235 ml/g, and solvent density was calculated through summation of the contribution of buffer components to 1.00658 g/cm3 at 4 °C using the program SEDNTERP (www.cauma.uthscsa.edu/software/). Molecular mass was determined using Beckman software provided as an add-on to Origin version 4.1. All nine data sets were analyzed using a global fit procedure based on a model describing an ideal non-interacting single component system, with local parameters for reference concentrations and base-line offsets and global parameters for the molecular weight. Best-fits were determined through visual inspection of the residuals. 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 × 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. Purification of IRF-3, IRF-3 5E, IRF-3 2A, and TBK1—FLAG-tagged IRF-3, the constitutively active mutant IRF-3 5E, the mutant IRF-3 2A (T404A/S405A), and the kinase TBK1 were purified from the supernatant of a freeze-thaw lysate of recombinant baculovirus-infected Sf21 cells using a FLAG resin followed by size exclusion chromatography on a Superdex S200 column. This procedure yielded about 0.6 mg of IRF-3 or IRF-3 2A, 0.3 mg of IRF-3 5E, and 0.9 mg of TBK1 per liter of insect cell culture. To analyze the effect of TBK1 phosphorylation on IRF-3 in vitro, we incubated TBK1 with IRF-3 or IRF-3 2A and analyzed the products by size exclusion chromatography. IRF-3 has the calculated molecular mass of 48 kDa. As shown in Fig. 1, purified IRF-3 eluted as a single peak with a Kav of 0.41, corresponding to an apparent molecular mass of 111 kDa. The mutant IRF-3 5E eluted as a single peak with a Kav of 0.24, corresponding to an apparent molecular mass of 316 kDa. When IRF-3 was preincubated with the kinase TBK1, the protein eluted in two peaks from the column. The first peak had a Kav of 0.41, the same as unphosphorylated IRF-3, and the second peak had a Kav of 0.24, identical to that of IRF-3 5E. SDS-PAGE analysis of the two peaks showed an upward mobility shift in the second peak, indicative of phosphorylation (data not shown). Thus TBK1 phosphorylation of IRF-3 leads to a higher molecular weight species with elution properties similar to those of the constitutively active mutant IRF-3 5E. The mutant IRF-3 2A exhibited identical elution profiles and responsiveness to TBKI phosphorylation as the wild-type protein (data not shown). 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. Mapping of the TBK1 Phosphorylation Sites—As shown in Fig. 1, TBK1 phosphorylation of IRF-3 results in two species, one of which elutes from a size exclusion chromatography column at the same position as dimeric IRF-3 5E. We concluded that TBK1 phosphorylation induces dimerization of IRF-3 in vitro, consistent with earlier findings. Having pure, properly folded, and biochemically homogeneous proteins allowed us to investigate further the role of phosphorylation in IRF-3 activation. We analyzed by mass spectroscopy tryptic peptides from unphosphorylated IRF-3, IRF-3 2A, IRF-3 5E, and TBKI-phosphorylated IRF-3 separated by size exclusion chromatography into the dimeric and monomeric fractions. The constitutively active and dimeric IRF-3 5E was 100% phosphorylated at a site 1 residue (Ser385 or Ser386; our data did not allow us to distinguish which serine). That is, site 1 had been phosphorylated by insect cell kinases prior to purification, and the protein had dimerized. This constitutive phosphorylation was observed only in the context of the 5E mutation, and there was no phosphorylation of insect cell-expressed wild-type IRF-3, all of which was monomeric. We do not know the identity of the relevant insect cell kinase. After phosphorylation by TBK1, all IRF-3 had acquired a phosphate at site 2 (Ser404 or Thr405; our data did not allow us to distinguish which residue). The IRF-3 that remained monomeric (peak at 13.6 ml in Fig. 1) had not been modified at site 1, whereas the IRF-3 that had dimerized (peak at 11.6 ml in Fig. 1) was fully phosphorylated at both sites 1 and 2. To investigate further the role of phosphorylation at Ser404 and Thr405, we mutated these residues to alanine (IRF-3 2A) and repeated the TBK1 kinase assay, gel filtration and phosphate mapping studies. The IRF-3 2A mutant dimerized, and peptide analysis showed that dimerization was dependent on phosphorylation of Ser385 or Ser386 as in the wild-type protein. In addition, we detected in both monomeric and dimeric TBK1-treated IRF-3 2A a single phosphorylation event in one of the three remaining site 2 positions (serine 396, 398, or 402). These data imply that phosphorylation at site 1 leads to dimerization, as both the TBK1 phosphorylation-dependent dimer and the IRF-3 5E dimer have been phosphorylated there. These data also show that phosphorylation at site 1 occurs only when site 2 bears a negative charge either from phosphorylation or from the 5E mutation. We never found a species that had a phosphate at site 1 without either a phosphate or a phosphomimetic at site 2. Finally, we can conclude that TBK1 phosphorylates preferentially at site 2, as this site was fully phosphorylated in both monomer and dimer, whereas only the fraction that had been converted to dimer had a phosphate at site 1 (Fig. 1). Our data indicate that positions in site 2 can be targeted redundantly by TBK1, as mutation of residues 404/405 to Ala did not abolish site 2 phosphorylation. Trypsin Digestion of IRF-3 and IRF-3 5E—The structures of the C-terminal activation domain of IRF-3 in the presence and absence of the IRF-3-binding domain from CBP have been determined previously (15Qin B.Y. Liu C. Lam S.S. Srinath H. Delston R. Correia J.J. Derynck R. Lin K. Nat. Struct. Biol. 2003; 10: 913-921Crossref PubMed Scopus (174) Google Scholar, 16Takahasi K. Suzuki N.N. Horiuchi M. Mori M. Suhara W. Okabe Y. Fukuhara Y. Terasawa H. Akira S. Fujita T. Inagaki F. Nat. Struct. Biol. 2003; 10: 922-927Crossref PubMed Scopus (129) Google Scholar, 19Qin B.Y. Liu C. Srinath H. Lam S.S. Correia J.J. Derynck R. Lin K. Structure (Camb.). 2005; 13: 1269-1277Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). They show that the conformations of the latent, autoinhibited IRF-3 and of the IBiD-bound complex are mutually exclusive. The autoinhibitory helices 1 and 5 must be displaced to expose a hydrophobic pocket containing the helices 3 and 4 that interact with the IBiD. Thus we expect that phosphorylation induces structural rearrangements in the domain, which ultimately allow interaction with IBiD and dimerization. We probed monomeric IRF-3 and dimeric IRF-3 5E by limited proteolysis (Fig. 3) and found that IRF-3 5E is more susceptible than wild type to tryptic digestion. After a 10-min incubation, most of the IRF-3 5E had been digested, whereas only a fraction of wild type had been cleaved at that time point. These data show that latent, autoinhibited IRF-3 is less labile than IRF-3 5E dimer. IRF-3 5E cleavage leads to two major products. N-terminal sequencing showed that the larger product derives from the N terminus of the protein and that the smaller product results from cleavage C-terminal to Arg194 (N-terminal LLVPGE). Limited proteolysis and mass spectrometry indicated that this fragment was further cleaved at residue 409, resulting in a stable proteolytic fragment comprising amino acids 195-409. Cleavage sites 195 and 409 are within autoinhibitory helices 1 and 5, respectively. In contrast, proteolytic cleavage of wild-type IRF-3 displayed" @default.
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• W2024592805 title "Interferon Regulatory Factor 3 Is Regulated by a Dual Phosphorylation-dependent Switch" @default.
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