IRAK4 Kinase Activation and Cytokine Induction 1 Interleukin 1/Toll-Like Receptor Induced Autophosphorylation Activates Interleukin 1 Receptor- Associated Kinase 4 and Controls Cytokine Induction in a Cell-Type Specific Manner**

IRAK4 is a central kinase in innate immunity but the role of its kinase activity is controversial. The mechanism of activation for IRAK4 is currently unknown; and little is known about the role of IRAK4 kinase in cytokine production, particularly in different human cell types. We show IRAK4 autophosphorylation occurs by an intermolecular reaction and that autophosphorylation is required for full catalytic activity of the kinase. Phosphorylation of any two of the residues T342, T345 and S346 are required for full activity and the death domain regulates the activation of IRAK4 . Using antibodies against activated IRAK4 we demonstrate that IRAK4 becomes phosphorylated in human cells following stimulation by IL-1R and TLR agonists, which can be blocked pharmacologically by a dual inhibitor of http://www.jbc.org/cgi/doi/10.1074/jbc.M113.544809 The latest version is at JBC Papers in Press. Published on February 24, 2014 as Manuscript M113.544809 Copyright 2014 by The American Society for Biochemistry and Molecular Biology, Inc. by gest on O cber 2, 2017 hp://w w w .jb.org/ D ow nladed from IRAK4 Kinase Activation and Cytokine Induction 2 IRAK4 and IRAK1. Interestingly, in dermal fibroblasts, while complete inhibition of IRAK4 kinase activity does not inhibit IL-1-induced IL-6 production, NF-kB or MAP kinase activation, there is complete ablation of these processes in IRAK4 deficient cells. In contrast, the inhibition of IRAK kinase activity in primary human monocytes reduces R848 induced IL-6 production with minimal effect on NF-kB or MAP kinase activation. Taken together, these studies define the mechanism of IRAK4 activation; and highlight the differential role of IRAK4 kinase activity in different human celltypes as well as the distinct roles IRAK4 scaffolding and kinase functions play. IRAK4 is the central kinase in the Tolllike receptor pathway of the innate immune system. Toll-like receptors constitute the first line of defense against pathogenic micro-organisms such as bacteria, viruses, and yeast. The TLR family also includes three cytokine receptors; the IL-1 receptor, the IL-18 receptor, and the IL-33 receptor. In addition to the innate immune system, IRAK4 is also expressed in T and B lymphocytes and non-hematopoietic/structural cells such as fibroblasts. In addition, IRAK4 regulates adaptive immunity and inflammatory responses mediated by TLR and IL-1 receptors. The TLR/IL-1 receptors all contain a cytoplasmic TIR domain which complexes with the bifunctional adaptor protein MyD88 that also contains a death-domain adaptor region. The death domain of MyD88 then recruits the kinases IRAK1 and IRAK4 into a signaling complex and associates with them via their death domains. It has been demonstrated that mice with a targeted deletion or a kinase-inactive version of IRAK4 are protected from disease in models of rheumatoid arthritis, atherosclerosis, multiple sclerosisand Alzheimer’s disease (1-5). Thus, IRAK4 is an enticing candidate for drug discovery of therapies for these diseases (1-5). The mechanism by which IRAK4 becomes activated is unclear. It is well known that certain kinases, e.g., members of the MAP kinase family, require inducible phosphorylation by an upstream kinase on their activation loop in order to activate (6,7). However, other kinases such as PKA and cGMP protein kinase require autophosphorylation to become fully activated (8,9). It has been shown that IRAK4 can undergo autophosphorylation in the presence of Mn 2+ -ATP (10). The sites within the activation loop that were identified by tandem mass spectroscopy in that study were T342, T345, and S346. The authors hypothesized that IRAK4 autophosphorylation is proceeded by an intramolecular mechanism because IRAK4 was not able to intermolecularly autophosphorylate in vitro in the presence of Mn 2+ ATP. In this study we examined the autophosphorylation of IRAK4 in the cell and in the presence of Mg 2+ , the physiological cation for ATP, in cell free enzymatic assays. In addition to confirming the autophosphorylation of T342, T345, and S346 we identified a fourth phosphorylation site, T352. We found that mutation of single residues to alanine did not significantly affect the catalytic activity of the protein, but that mutations of dual combinations of residues T342, T345, and S346 completely abolished activity. These data suggest autophosphorylation of IRAK4 leads to the activation of its kinase activity. We show that autophosphorylation of these activation loop residues are inducible upon treatment with R848 in primary human monocytes and IL-1β in human dermal fibroblasts and that this autophosphorylation proceeds via an intermolecular mechanism both in the enzymatic and in the cellular context. Additionally, we demonstrate that the kinase domain of IRAK4 is constitutively phosphorylated in the cell but the full-length kinase only becomes phosphorylated following stimulation. This demonstrates the role of the death domain both in maintaining the kinase in an inactive state and in the induction of the kinase activity. Importantly, we also show that pharmacologic inhibition of IRAK4 by an IRAK4/IRAK1 dual inhibitor completely blocks IRAK4 autophosphorylation but, surprisingly, has minimal effects on activation of NF-kB, p38, JNK, and ERK in both human dermal fibroblasts and primary human monocytes. We find, as reported previously, that human dermal fibroblasts from patients with autosomal recessive IRAK4 deficiency do not activate NF-kB, p38, JNK, and ERK, and do not produce cytokines in response to IL-1β (11-15). SV40 transformed dermal by gest on O cber 2, 2017 hp://w w w .jb.org/ D ow nladed from IRAK4 Kinase Activation and Cytokine Induction 3 fibroblasts from IRAK4 deficient patient 15 (IRAK4-/-) and a healthy control (wild-type) were used (12). Interestingly, we observed that the inhibition of IRAK4 autophosphorylation blocks cytokine production in primary monocytes but not in dermal fibroblasts. These data clearly demonstrate that there are different roles of IRAK4 kinase activity and scaffolding activity in different human cell types. EXPERIMENTAL PROCEDURES: Cloning and expression of IRAK4: The full-length ORF of IRAK4 (Genbank number AF445802) was obtained from Invitrogen. Both the full-length and the kinase domain (residues 154-460) were cloned with the addition of either C-terminal Flag tags or 6xHis tags via PCR into the Gateway entry vector pDONR 201 (Invitrogen) according to the manufacturer’s instructions. For eukaryotic cell expression, the Cterminal flag-tagged constructs were recombined into the Gateway expression vector pcDNA3DEST40 (Invitrogen). Mutagenesis was performed via PCR using KOD polymerase as previously described (16). Human dermal fibroblast or HEK 293T cells were transfected with Lipofectamine 2000 (Invitrogen) according to the manufacturer’s directions. For baculovirus expression, C-terminal 6xHis tagged constructs were recombined into the baculovirus expression vector pDEST8 and baculovirus was produced via the Invitrogen baculovirus system. For expression, Sf21 cells were infected with virus at an MOI of 1:1 and grown for 48 hours in Invitrogen InsectGro media at 27C. Protein Purification: Sf 21 cells expressing IRAK4 full-length protein or kinase domain were lysed via Paar Bomb at 4 oC in lysis buffer (50 mM Phosphate pH 8, 300 mM NaCl, 25 mM Glycerophosphate, 10 mM NaF, 1 mM Orthovanadate, 5 mM beta mercaptoethanol and EDTA Free Protease Inhibitors (MERK). The kinase domain was initially purified via Ni affinity chromatography (Ni NTA, Qiagen) and contaminates removed by flow through cation exchange chromatography (Poros HS, Applied Biosystems). The protein was captured via anion exchange chromatography (Poros HQ, Applied Biosystems) and eluted with a 15 CV salt gradient (buffer A: 20 mM HEPES pH 7.5, 5 mM DTT, 50 mM NaCl, Buffer B: 20 mM HEPES pH 7.5, 5 mM DTT, 1M NaCl). The protein was then brought to greater than 99% purity via gel filtration chromatography (Tosoh G3000SW column). Separation and Characterization of IRAK4 Protein Phosphorylation States: The different phosphorylation forms of the protein were separated via high affinity strong anion exchange chromatography on a Mono Q column (GE Healthcare) using a very shallow gradient of 40 column volumes (buffer A: 20 mM HEPES pH 7.5, 5 mM DTT, 50 mM NaCl, Buffer B: 20 mM HEPES pH 7.5, 5 mM DTT, 600 mM NaCl). Autophosphorylation of IRAK4 kinase domain was shown using purified kinase domain with and without dephosphorylation by lambda phosphatase (New England Biolabs). Purified kinase domain was allowed to react with 10 mM MgCl2 and 1 mM ATP for 1 hour at RT. The varied phosphorylation states were then purified via high affinity strong anion exchange chromatography and analyzed by LCMS. Measurement of initial rates: The enzymatic rate was determined at 25 o C using the coupled pyruvate kinase (PK)/lactate dehydrogenase (LDH) assay, at 340 nm on a Molecular Devices plate reader as previously described (17). The reaction was carried out in a final volume of 80 uL, in 25 mM HEPES (pH=7.5), 10 mM MgCl2, 2 mM DTT, 0.008% Triton X-100, 100 mM NaCl, 20 units of PK, 30 units of LDH, 0.025 mM NAD, 2 mM phosphoenol pyruvate, and phosphorylated or dephosphorylated forms of IRAK4 kinase domain or the full-length construct at 50 nM (17). In vitro assay for IRAK4 activity of mutants: Flag tagged IRAK4 kinase domain mutant proteins were isolated from HEK 293T cells following transfection with Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Cells were lysed in Triton lysis buffer (1% Triton-X100, 20 mM HEPES pH=7.4, 100 mM NaCl) containing protease inhibitors (Sigma chemical) at 4 C. Cell lysates were immunoprecipitated with anti-flag M2 beads (Sigma) washed extensively in lysis buffer and eluted with 200 μg/mL Flag peptide (Sigma Chemical) in 10 mM HEPES pH=7.4 with 150 mM NaCl. Eluates were dialyzed at 1:1000 against 20 mM Tris-HCl pH= 7.5, 150 mM NaCl to remove Flag peptide. Protein was diluted to10 by gest on O cber 2, 2017 hp://w w w .jb.org/ D ow nladed from IRAK4 Kinase Activation and Cytokine Induction 4 ng/mL stock and assayed for activity by a DELFIA assay. Briefly, 10 ng/mL IRAK4 protein was pre-incubated with 2 mM ATP in a kinase reaction buffer of 20 mM HEPES pH=7.5, 5 mM MgCl2, 0.0025% Brij for 1 hr at 25 C. This protein was diluted to 5 nM and incubated with 200 nM peptide (biotinylatedAGAGRDKYKTLRQIR, Tufts University) with 2 mM ATP for 1 hour in reaction buffer. The reaction was stopped by the addition of 20 mM EDTA. 100 uL of reactions were transferred to a 96 well streptavidin coated plate (R&D Systems) and allowed to bind for 30 minutes followed by extensive washing with 1xTBS containing 0.02% Tween. Plates were then incubated with antiphospho Ezrin/Radixin/Moesin antibody (Cell Signaling Technology) diluted 1:1000 in blocking buffer (10 mM MOPS pH=7.5, 150 mM NaCl, 3 mM NaN3, 0.025%Tween 20, 0.2% gelatin, 2% BSA) for 1 hour followed by extensive washing with TBS/Tween. Plates were developed by incubation for 30 minutes with an anti-rabbit IgG – europium conjugate (Perkin-Elmer) diluted 1:5000 in blocking buffer followed by extensive washing in TBS/Tween and finally the addition of DELFIA enhancement solution (Perkin-Elmer). Plates were read in an Envision reader (PerkinElmer) on a Europium setting. Antibodies: Polyclonal IRAK4 antibodies were made by contract with Biosource Inc against a C-terminal peptide of human IRAK4 (CEKTIEDYIDKKMNDADSC) or were purchased from Cell Signaling Technology. Monoclonal antibodies to IRAK4 were obtained from Abnova Corporation. Antibodies to total and phospho-p38, total and phospho-JNK, MyD88, and human IRAK1 were purchased from Cell Signaling Technology. Phosphospecific antibodies to sites on IRAK4 (pT342, pT345, pS346, pT352, pT345/pS346, pT342/pT345, pT342/pT346) were made by contract with 21 st Century Biochemicals (Marlborough, MA). Antibodies were immunodepeleted against non-phosphorylated peptides and, in the case of antibodies against dual phosphorylated sites, immunodepleted against both monophosphorylated peptides in order to ensure specificity. In vitro intermolecular phosphorylation assay: Flag–tagged kinase-inactive form (D329A) of the kinase domain of IRAK4 was expressed and purified as described above in “In vitro assay for IRAK4 activity of mutants”. Immunoprecipitated kinase-inactive proteins were incubated with 20 nM of fully phosphorylated IRAK4 full-length or kinase-domain proteins purified from baculovirus in kinase reaction buffer containing 2 mM ATP for 1 hour. Reactions were analyzed by western blotting with total IRAK4 and anti-pT345pS346. Transfection of human dermal fibroblasts and immunoprecipitation of mutant proteins: SV40 transformed dermal fibroblasts from IRAK4 deficient patient 15 IRAK4-SV40 fibroblast: 11096_40+23del/1-1096_40+23del. (IRAK4-/-) and a healthy control (wild-type) provided by Jean-Laurent Casanova were used (12). Cells were grown to 80% confluence in DME + 10% FBS and transfected with wild-type and mutant IRAK4 constructs using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s directions. Generally 5 μg of construct in pcDNA3-DEST40 vector (Invitrogen) was used for a 10 cm plate. Transfected cells were placed in DME+0.5% FBS 2 hours prior to stimulation. Cells were stimulated with 10 ng/mL IL-1β (R&D Systems) for 30 minutes and then lysed in 1 mL of Triton lysis buffer (above) containing protease inhibitors, 1 mM Na-vanadate, and 5 mM βglyceroposphate. Recombinant IRAK4 proteins were immunoprecipitated using anti-flag M2 sepharose beads (Sigma) Monocyte enrichment and treatment: Buffy coats (from MGH) were incubated with RosetteSep Cocktail (Stem Cell Technologies) and purified according to the manufacturer’s directions. Monocytes were resuspended for culture in RPMI + 0.5% FBS. Monocytes were then pretreated for 30 minutes with 10 uM Compound 1 and then stimulated with 1ug/ml TLR7/8 agonist R848 for 15 minutes (phosphoprotein) and 4h (cytokines). Dermal Fibroblast Treatment: Wild-type and IRAK4-null human dermal fibroblast cells (11) were grown to 80% confluence and then pretreated for 30 minutes with 10 μM Compound 1 followed by stimulation with 10ng/ml IL-1β and collected post-stimulation at 15 minutes (phosphoprotein) and 4h (cytokines). Western Blotting: Samples were washed with ice cold PBS and lysed in 200 ul protein lysis buffer (Novex), sonicated, heated to 95C for 2 minutes and then separated by SDSpolyacrylamide gel electrophoresis and transferred by gest on O cber 2, 2017 hp://w w w .jb.org/ D ow nladed from IRAK4 Kinase Activation and Cytokine Induction 5 to nitrocellulose membranes. Membranes were then blocked in Odyssey Blocking Buffer (Li-Cor) and incubated overnight in primary antibody at 4C. Membranes were then washed 3X for 5 minutes each in 1X PBS-T and incubated in secondary antibody for 60 minutes at RT. Membranes were again washed 3X for 5 minutes each in PBS-T and imaged on the Odyssey CLx (Li-Cor). ELISA: Culture supernatants were collected 1 and 4h after stimulation, aliquoted to 4-plex pro-inflammatory II plates (MSD N45025B-1) and developed according to the manufacturer’s directions. ActivX analysis of human monocytes: 50 million human monocytes were incubated with 10 μM Compound 1 for 30 minutes and 50 million with DMSO. Both groups were then spun down at 1500 rpm in a Sorvall Legend RT swinging bucket centrifuge for 10 minutes and frozen on dry ice. The cell pellet was then sent to ActivX Biosciences (La Jolla CA) for analysis in the KiNativ in situ kinase profiling panel. RESULTS: Expression of IRAK4 and separation of distinct phosphorylation states by ion-exchange chromatography: The C-terminal 6xhis tagged kinase domain (aa 154-460) and the full-length construct of IRAK4 were expressed in Sf21 cells. Chromatography via size-exclusion showed a single peak at 35 kDa for the kinase-domain construct. We observed that Ni-NTA chromatography of the full-length protein gave two species of IRAK4 – the full-length protein and a smaller, 34 kDa fragment (data not shown). This smaller fragment could be readily removed by size exclusion chromatography to give a homogeneous preparation of the full-length protein. Five milligrams of IRAK4 kinase-domain protein from Ni-NTA were bound to a column of Q-Sepharose and eluted via a salt gradient from 50 mM to 1 M NaCl over 40 column volumes. Two distinct peaks were observed that could be interconverted by incubation with 1 mM ATP or with γ-phosphatase demonstrating that the mobility shift is due to phosphorylation (data not shown). ESI-LC MS on the individual peaks revealed that the faster migrating peakhad a molecular weight of 35,383 Da. This differs from the expected molecular weight for the IRAK4kinase domain construct of 35,330 Da by 50 Da indicating the possibility of N-terminal acetylation. Similar analysis of the slower migrating peak showed a molecular weight of 35,622, which correlates to the presence of an Nterminal acetylation and 3 phosphates (data not shown). A much smaller amount of the phosphoprotein from this peak had a molecular mass of 35,700 which contained 4 phosphates with the N-terminal acetylation. Characterization of phosphorylation sites on IRAK4 kinase domain via HPLC ESI-MS-MS: In order to unambiguously determine the major sites of phosphorylation on the fully phosphorylated form of the IRAK4 kinase domain, the IRAK4 kinase domain autophosphorylated protein was subjected to tandem mass spectrometric analysis following trypsin digestion. Four distinct peptides were observed: T3 (residues 165-174 (positions denoted from the N-terminal of the full-length protein), T4 (residues 175-191), T19 (residues 339-347) and T20 (residues 348362) (Supp. Fig. S1). Ion chromatograms of these peptides showed that the T3, T19 and T20 peptides are all phosphorylated. Further separation and analysis showed that T3 was weakly phosphorylated on S167 and that T20 was phosphorylated on T352. Both peptides contained only one phosphorylation site. The T19 peptide had phosphorylation sites at T342, T345, and S346 (Supp. Fig. S1). While this peptide appeared as three distinct singly phosphorylated peptides it also appeared as three distinct dual phosphorylated peptides. The dual phosphorylated peptides eluted were of three possible combinations: pT342/pT345, pT342/pS346, or pT345/pS346 (Supp. Fig. S2). Therefore, our results indicate that the majority of triphosporylated species consist of at least one of the dual phosphorylated species on peptide T19 in combination with T352. The identified phosphorylation sites in the kinase domain are shown within the context of the full protein sequence in Fig. 1A and the IRAK4 structure with phosphorylation sites in Fig.1B. Cheng et al. (10) observed phosphorylation in the active site on T342, T345, and S346 but not on T352. This difference could be due to the fact Cheng et al. (10) used Mn 2+ as the cationic metal to co-ordinate the ATP whereas we used Mg 2+ , which is the physiological metal. by gest on O cber 2, 2017 hp://w w w .jb.org/ D ow nladed from IRAK4 Kinase Activation and Cytokine Induction 6 The difference could also be due to the fact that the phosphorylation sites that we identified came from a constitutively autophosphorylated kinase domain whereas Cheng et al (10) used the fulllength protein. Activity of phosphorylated and nonphosphorylated forms of IRAK4: It is well known that autophosphorylation of certain kinases can increase their activity (9,18). To qualitatively determine if there is a difference in activity between the phosphorylated and nonphosphorylated forms of full-length IRAK4 and its kinase domain, we observed the initial rates of conversion of a substrate peptide via a coupled enzyme assay. As shown in Fig. 2A, the phosphorylated form of both the kinase-domain and the full-length protein show a consistent and constant linear rate of ATP conversion. However, the non-phosphorylated forms of the protein demonstrate a lag phase, where the initial rate is slow but gradually increases over time and eventually reaches the same linear rate as the phosphorylated form. These results suggest that the fully phosphorylated form of the protein is more catalytically active than the nonphosphorylated form. They also suggest that the change in rate over time of the nonphosphorylated form is due to autophosphorylation, which increases the catalytic activity. In addition, the length of the “lag” phase was reduced as the concentration of the nonphosphorylated full-length protein increased (data not shown). The concentration dependence of the lag phase and of the rate indicates a second order reaction and demonstrates that the phosphorylation step is most likely intermolecular. We also observed that the presence or absence of the death domain did not affect the rate of autophosphorylation, the rate of reaction, or the length of the lag-phase as the rates of ATP conversion are similar (Fig. 2A). These data demonstrate that IRAK4 kinase is constitutively active in the presence and absence of the death domain in a cell free reaction. Activity of activation loop mutants in the IRAK4 kinase domain: In order to determine the contribution of the putative phosphorylation sites to IRAK4 activity, single mutations to alanine at positions 342, 345, 346, and 352 were made in the C-terminal flag tagged kinase domain construct and expressed in HEK 293T cells. Several combinations of mutations at these residues were also made and evaluated. The protein was purified to >70% purity and the enzymatic activity of the purified mutants was determined at 1 mM ATP/10 mM MgCl2 following an 1 hour pre-incubation in this concentration of ATP/MgCl2 to maximize the level of autophosphorylation to ensure a constant initial rate of kinase activity. The activity was determined via fluorescent ELISA (DELFIA) assay as described in “Experimental Procedures”. The activity of the mutants was in the linear range of a standard curve determined with wild-type kinase and normalized to the protein concentration by western blot and Coomassie blue staining. As shown in Fig. 2B we observed that the single mutations of any of these residues to alanine did not substantially inhibit the enzymatic activity. However, dual and triple combinations of mutations at T342, T345, and S436 substantially inhibited activity, indicating that at least two of these residues must be phosphorylated for activity. This finding is in contrast to the findings of Cheng et al (10) who found that single mutations to alanine at T342, T345, or S346 substantially inhibited the activity of the kinase. The single mutation of T352 to alanine did not significantly affect the activity nor did the mutation of T352 in combination with single mutations of T342, T345 or S346 (data not shown). Therefore, phosphorylation of T352 is not essential for kinase activity. The physiological importance of phosphorylation of T352 is unclear, however, it is observed in the crystal structure of IRAK4 that the hydroxyl side chain of T352 is exposed to solvent and lines the substrate binding pocket as shown in Fig.1B (19). It is possible that a phosphate at T352 could induce a different binding position for the substrate and allow phosphorylation of different residues on the substrate or allow for phosphorylation of a different substrate protein. It should be noted that we were unable to show phosphorylation of T352 in cells using our antibody to pT352 (data not shown). Thus, the physiological significance of this phosphorylation remains to be determined. In order to develop reagents to observe specific phosphorylated states of IRAK4 and determine their physiological significance, we made phosphospecific antibodies to pT342, pT345, pS346, pT352 and to the dual phosphorylated species of pT342/pT345, by gest on O cber 2, 2017 hp://w w w .jb.org/ D ow nladed from IRAK4 Kinase Activation and Cytokine Induction 7 pT342/pS346, and pT345/pS346. The antibodies to the monophosphorylated sites were immunodepleted over the corresponding nonphosphorylated peptides, and in the case of the dual phosphorylated species, over each monophosphopeptide as well. This ensured that the antibodies will specifically recognize the requisite phosphorylated species. The antibodies did not recognize the purified unphosphorylated form of full-length IRAK4 but robustly recognize the purified autophosphorylated form of the fulllength IRAK4 in western blots (data not shown) This data recapitulated the results of the mass spectrometry and show that these residues are indeed autophosphorylated in the intact protein at all of the identified residues in vitro. The most robust signal was obtained with the antibody against pT345/pS346, henceforth referred to as the pIRAK4 antibody IRAK4 can be autophosphorylated intermolecularly on activation loop residues in vitro: Our observation that the initial lag phase in kinase activity is dependent on concentration strongly suggests that IRAK4 autophosphorylates by an intermolecular mechanism. Previously, Cheng et al (10), showed that IRAK4 does not autophosphorylate via an intermolecular reaction. Their experiment monitored the ability of wildtype kinase to phosphorylate the kinase-inactive mutant at 30μM ATP with 2 mM MgCl2 and detection was monitored using a phosphospecific antibody to pT345pS346. We performed a similar experiment at a more physiological condition using 2 mM ATP and 5 mM MgCl2. We monitored the ability of the active full-length kinase to phosphorylate the kinase-inactive D329A kinase domain protein. As shown in Fig. 3, the kinase-inactive kinase domain was phosphorylated at pT345/pS346, clearly demonstrating that these residues can be phosphorylated via an intermolecular mechanism. Additionally, we observed a slight mobility shift upon autophosphorylation of the full-length kinase domain which was partially inhibited by the kinase-inactive D329A kinase domain (Fig. 3). Our results show that mammalian IRAK4 becomes activated by a similar mechanism as Pelle, the Drosophila homolog of IRAK4, which has been shown to be activated by intermolecular autophosphorylation (20). IL-1-induced autophosphorylation of IRAK4 in human dermal fibroblasts: We transfected with flag-tagged wild-type IRAK4 or kinase-inactive (D329A) mutant and treated with IL-1β for 30 minutes. Immunoprecipitation with anti-flag antibody was followed by western blotting with the pIRAK4 (pT345pS346) antibody. We observed robust IL-1βinduced phosphorylation at the dual site T345/S346 (Fig. 4A). We were unable to detect phosphorylation on monophosphorylated sites or other dual phosphorylated sites in the over-expressed protein. This could be due to low levels of phosphorylation at these sites by IL-1 activation or, alternatively, to low affinity of these antibodies for their epitopes. We repeated this experiment with the kinaseinactive mutant D329A which is unable to autophosphorylate at T345/S346 in vitro. Surprisingly, after western blotting of immunoprecipitated over-expressed D329A from IL-1 treated dermal fibroblast cells with pIRAK4 pT345/pS346 antibody, we observed that the D329A mutant of IRAK4 was also inducibly phosphorylated on these residues similar to the wild-type protein (Fig. 4A). This demonstrates that IL-1 induced phosphorylation of the activation loop of IRAK4 can come from transphosphorylation by active endogenous IRAK4 or by an upstream kinase. Loss of endogenous IRAK4 prevents IL-1 inducible phosphorylation of ectopically expressed IRAK4: In order to determine if IRAK4 phosphorylation is mediated by intermolecular IRAK4 autophosphorylation or another independent kinase, we determined whether kinase-dead IRAK4 kinase can be phosphorylated in the IRAK4 deficient dermal fibroblast cells. As shown in Fig. 4B, the IL-1 induced autophosphorylation of the kinase-inactive IRAK4 is preserved in the wild-type cell line but it does not occur in the IRAK4 deficient cell line. This result clearly shows that, within the cell, another molecule of IRAK4 is responsible for the phosphorylation and activation of IRAK4. The death domain prevents constitutive activation of IRAK4 kinase activity: The death domain of IRAK4 is believed to be integral for the recruitment of the IRAK kinases to the receptor/MyD88 signaling complex. To assess whether autophosphorylation of T345/S346 is dependent on the presence of an intact death by gest on O cber 2, 2017 hp://w w w .jb.org/ D ow nladed from IRAK4 Kinase Activation and Cytokine Induction 8 domain, we transfected dermal fibroblast cells with the flag-tagged wild-type kinase domain only, as well as a kinase-inactive form of IRAK4 (D329A) kinase domain. We observed robust IL-1 inducible phosphorylation of endogenous IRAK4 in the whole cell lysate (Fig. 5A). We observed a band in the pIRAK4 blot at the molecular weight of the transfected wild-type kinase domain (~35 kDa) in the unstimulated cells which was not present in the vector or kinaseinactive lanes. This data suggest that a significant amount of constitutive autophosphorylation occurs on the wild-type IRAK4 kinase domain in unstimulated cells. It is worth noting that the pIRAK4 blots also detected a 35 kDa band in the IL-1β treated cells, likely due to the proteolytic fragment from the endogenous IRAK4 which was also observed by Hatao and colleagues following stimulation of macrophages with TLR agonists (21). In order to directly look at the phosphorylation of transfected IRAK4 kinase domain, we performed the immunoprecipitation with an anti-flag antibody followed by immunoblot with the pIRAK4 antibody. In this experiment, the constitutive autophosphorylation of the wild-type but not the kinase-inactive form of the kinase domain becomes readily apparent. (Fig. 5B) Unlike the endogenous full-length IRAK4, the wild-type kinase domain was constitutively phosphorylated in the absence of IL-1β and the phosphorylation level did not increase after treatment with IL-1β. Furthermore, the D329A kinase domain mutant, unlike the full-length D329A mutant, did not show any phosphorylation at T345/S346 in either the presence or absence of IL-1β (Fig. 5B). It is clear from these results that the ability of IL-1 to induce phosphorylation of IRAK4 is dependent on an intact death domain. The death domain acts as a scaffold to recruit the kinase to the receptor associated MyD88. Thus, the inability of kinase-inactive kinase domain to be phosphorylated could be due to the inability of it to be recruited to the receptor associated MyD88 and another molecule of endogenous IRAK4. Importantly, the fact that the active kinase domain is constitutively phosphorylated in the absence of the death domain also implies that the death domain plays a role in keeping the full-length protein inactive in the absence of activation by IL1R or TLRs. Thus the death domain may play a role as a negative regulator of IRAK4 kinase activity in the context of the unstimulated cell. Pharmacological inhibition of IRAK4 kinase blocks IRAK4 phosphorylation in both human monocytes and dermal fibroblasts: Having demonstrated that autophosphorylation of IRAK4 occurs physiologically, we were interested in using phosphorylated IRAK4 as a potential biomarker to determine the extent of inhibition of IRAK4 activation in cells using a pharmacological inhibitor. We wanted to see if pharmacological inhibition of IRAK4 kinase would inhibit phosphorylation of IRAK4 in the context of the TLR/IL-1R pathway and what functional consequences this inhibition may have in different human cell types. We utilized dermal fibroblasts due to their availability from wild-type and IRAK4 deficient patients. We also examined primary human monocytes from healthy volunteers, since we were interested in innate immune cells where IRAK4 activity plays an important role as an inflammatory mediator. We used Compound 1, a previously characterized inhibitor of IRAK4 kinase for our studies on the pharmacology of IRAK4 in human cells (19). This compound is highly potent against IRAK4 (IC50=2 nM at Km=300 uM ATP)(22) and to a lesser extent IRAK1 (IC50=15 nM at Km=35 uM ATP). Though it possesses excellent selectivity, it has poor physical properties such as solubility and permeability, which necessitated use of micromolar concentrations for our studies. In order to test target occupancy in the context of the cell, we subjected cells treated with 10 uM of Compound 1 to analysis by ActivX technology in the Kinativ panel (23). We observed that the compound has excellent selectivity against 170 kinases in the panel with 90% occupancy against IRAK4 and only 55% occupancy against IRAK1. The only other kinases that were inhibited greater than 70% were PIP4K2C (76%), and Aurora A and B (71%), none of which are known to inhibit TLR/IL-1R signaling (Supp. Table ST1). We used the TLR7/8 agonist R848 to activate IRAK4 in human monocytes and IL-1β to activate IRAK4 in wild-type and IRAK4 deficient dermal fibroblasts. The choice of agonist is based on the ability of these agonists to induce cytokine production in these cell types. Pharmacological inhibition of IRAK4 kinase with 10 uM of Compound 1 completely blocks IRAK4 by gest on O cber 2, 2017 hp://w w w .jb.org/ D ow nladed from IRAK4 Kinase Activation and Cytokine Induction 9 autophosphorylation in dermal fibroblasts. Compound 1 also significantly but does not completely block ubiquitin modification of IRAK1, demonstrating that post-translational modification of IRAK1 is not entirely dependent on IRAK4 kinase activity. This is in contrast to IRAK4 deficient fibroblasts where IL-1β induced modification of IRAK1 is completely absent (Fig. 6A). Compound 1 also completely blocks IRAK4 phosphorylation in human monocytes (Fig. 6B). IRAK4 kinase activity is required for cytokine production in human monocytes but dispensable in dermal fibroblasts: We next wanted to investigate the downstream functional consequences of this block in IRAK4 phosphorylation. Using the pharmacological inhibitor of IRAK4 kinase in both human monocytes and human dermal fibroblasts we measured IL-1, IL-6 and TNF-α cytokine induction. Fig. 7 shows that these cytokines are significantly reduced with the IRAK4 inhibitor in human monocytes, however there is no effect on cytokine production in the wild-type dermal fibroblasts. As expected, there is no induction of cytokines in the IRAK4 deficient dermal fibroblasts. The data suggest that in monocytes, the kinase activity of IRAK4 is required for cytokine production. However, in dermal fibroblasts, while deficiency of IRAK4 abolishes cytokine production entirely, inhibition of kinase has no effect on these cytokines. This result is consistent with a previous report showing that reconstitution of IRAK4 deficient fibroblasts with kinaseinactive full-length IRAK4 can reconstitute IL-1 induced IL-6 production (24). Thus, IRAK4 protein scaffold activity is required but not the kinase activity in dermal fibroblasts, suggesting IRAK4 scaffolding activity allows cytokine production to continue in this cell type. Clearly, there are different roles for IRAK4 kinase activity and scaffolding activity in different cell types. Minimal effects on NF-kB and MAPK are observed with complete inhibition of IRAK4 kinase in dermal fibroblasts and monocytes: Normal TLR/IL-1R signaling can exert its effects on cytokines via activation of NF-kB and MAPKs including the core MAPK pathways, JNK, ERK and p38. Therefore, we investigated the activation status of these pathways. Surprisingly, our data revealed only minimal inhibition of pp38, pERK, pJNK and pp65 in both cell types following complete inhibition of IRAK4 kinase (Fig. 8). Understanding the exact signaling events mediated by IRAK4 kinase activity that lead to cytokine production in monocytes, as well as the scaffold function of IRAK4 in dermal fibroblasts that is responsible for cytokine production, is the focus of our immediate future studies. DISCUSSION: We have identified four phosphorylation sites within the activation loop that are the result of autophosphorylation: T342, T345, S346, and T352. Previously, Cheng et al (10) identified T342, T345, and S346 by MS analysis and other groups identified these residues by crystallography (19,22). In this report we present several lines of evidence to support the hypothesis that autophosphorylation of IRAK4 is required for the full activation and subsequent signaling events in a cell-type specific manner. First, we showed purified unphosphorylated IRAK4 exhibited a lag in the kinase assay in contrast to the phosphorylated form, suggesting the requirement of autophosphorylation for the kinase activity. Second, we show that mutation of at least two residues at positions T342, T345, and S346 reduce the kinase activity. These data suggest that phosphorylation at these sites are required for the full activation of IRAK4 kinase activity. Third, importantly, inhibition of IRAK4 phosphorylation can be achieved by an IRAK4/1 inhibitor and results in the reduction of cytokine production in a cell-type specific manner, i.e. reduction in human monocytes but no effect in dermal fibroblasts. Unphosphorylated full-length IRAK4 can autophosphorylate in vitro in a cell-free system. In the cellular context, however, autophosphorylation is inducible by IL-1β. The mechanism of intermolecular autophosphorylation is inferred from the observations that wild-type IRAK4 can phosphorylate the kinase-inactive construct in an in vitro assay, that autophosphorylation in vitro is concentration dependent, and that over-expressed kinase-inactive IRAK4 is inducibly autophosphorylated in the wild-type but not in an IRAK4 deficient cell line. The inducibility of IRAK4 autophosphorylation in the cellular context is dependent on the death domain. This is demonstrated by the fact that both the full-length form of kinase-active and kinaseby gest on O cber 2, 2017 hp://w w w .jb.org/ D ow nladed from IRAK4 Kinase Activation and Cytokine Induction 10 inactive IRAK4 are inducibly phosphorylated following stimulation with IL-1β in wild-type human dermal fibroblasts. In contrast, kinasedomain constructs exhibit different behavior from the full-length constructs under the same conditions. Kinase-active constructs of the IRAK4 kinase domain constitutively autophosphorylate when expressed in wild-type human dermal fibroblasts in the absence of IL-1β stimulation but the kinase-inactive construct of the kinase domain are not phosphorylated in either the presence or absence of IL-1β stimulation. This data clearly demonstrates the importance of the death domain in regulating the kinase activity of IRAK4 in the cellular context. It is likely that IRAK4 becomes activated because ligand binding by the IL-1/TLR receptor causes the aggregation/multimerization of receptors which recruits the adaptor protein MyD88 via the TIR domains of the receptor and MyD88. This complex in turn recruits molecules of IRAK4 via the death-domains of IRAK4 and MyD88 into the signaling complex. The high local concentration of IRAK4 in this complex promotes intermolecular autophosphorylation and activation of the kinase. Indeed, it has been shown that the death domain of MyD88 and the deathdomain of IRAK4 form multimeric complexes in vitro (25,26). Thus, based on this model, the kinase-inactive form of the kinase domain does not become autophosphorylated because it lacks the death domain and is unable to associate with MyD88 and undergo intermolecular phosphorylation by the endogenous IRAK4 as observed in Fig. 5B. This mechanism of activation is reminiscent of that proposed for Pelle, the Drosophila analog of the IRAKs (20,27). In this model, Pelle and the adaptor protein Tube are preassociated with the receptor Toll. Upon binding of the ligand Spatzle, the Toll receptors dimerize causing intermolecular autophosphorylation of Pelle and the subsequent activation of signaling. The high degree of homology between Pelle and human IRAK proteins support a similar activation mechanism (28). Another possible model of activation is one in which the death domain of IRAK4 associates with a protein that keeps IRAK4 inactive until it is released by receptor activation. The fact that both the kinase domain and the fulllength protein are able to autophosphorylate in in vitro assays with similar rates (Fig. 2A) indicates that the death domain alone is insufficient to keep the kinase inactive. This implies that the death domain or its interacting protein in the cellular context is keeping the protein inactive until it is released upon stimulation by IL-1R or TLR ligands. A similar function has been proposed for the protein Tollip which has been found to have a negative regulatory effect on IRAK1 (29). Importantly, our data using the IRAK4 inhibitor Compound 1 also support that IRAK4 phosphorylation is an autophosphorylation event and is associated with cytokine induction in a celltype specific manner. The inhibitor used has an IC50 for IRAK4 of 2 nM and for IRAK1 of 15 nM (19), making it difficult to definitively assess the contribution of either kinase on cellular signaling. However, assessment of ATP binding site occupancy by the inhibitor in the cell shows that at the concentrations tested (10 uM), IRAK4 is 90% occupied whereas IRAK1 is only 55% occupied by the inhibitor, indicating that most of the effects are derived from inhibition of IRAK4 (see Supp. Table ST1). However a more selective inhibitor of IRAK4 over IRAK1 will be required to clearly define the role of IRAK4 kinase activity. We show that the inhibitor completely blocks IRAK4 phosphorylation but has minimal effects on activation of NF-kB, p38, JNK and ERK in IL-1R or TLR7/8 stimulated human dermal fibroblasts and monocytes. The inhibitor does not block IL-6 and TNF-α production in human dermal fibroblasts whereas these cytokines are blocked in human monocytes, suggesting celltype specific requirements for IRAK4 kinase activity in human cells. Our data are not consistent with that of Chiang, et al. (30) who showed no effect of an IRAK4/IRAK1 dual inhibitor on cytokine production in human monocytes. It is possible that the inhibitor used by Chiang et al. was not potent enough to inhibit IRAK4 kinase activity as there was no measurement of pIRAK4 to gauge the extent of inhibition in the monocytes. Indeed, the potency of the inhibitor described by Chiang et al. (30) was considerably less potent against IRAK4 and IRAK1 than the inhibitor used in this study and we have been unable to document inhibition of pIRAK4 in either IL-1β stimulated dermal fibroblasts or R848 stimulated primary human by gest on O cber 2, 2017 hp://w w w .jb.org/ D ow nladed from IRAK4 Kinase Activation and Cytokine Induction 11 monocytes at the concentrations described in that paper (data not shown). The contribution of IRAK4 catalytic versus scaffold activity in regulating cytokine production is shown to be cell-type specific in human cells. In dermal fibroblasts, our data suggest IRAK4 utilizes its scaffolding activity to allow signaling and cytokine production to go forward unhindered in the absence of kinase activity. However, in monocytes, the kinase activity is required for cytokine production. This suggests cell-type specific requirements for IRAK4 scaffolding versus kinase activity consistent with previous reports (24,30,31). More detailed studies need to be undertaken on cell-type differences in the requirement for IRAK4 kinase. The minimal effect of an IRAK4 inhibitor on MAP kinase and NF-kB activation in both cell types is consistent with the study using macrophages from IRAK4 kinase-inactive knockin mice (32,33). These data suggest that IRAK4 kinase activity may impact a novel, yet to be determined pathway in regulating cytokine production. Perhaps, as suggested, the kinase activity may impact cytokines via targeting the mRNA stability of genes containing AU-rich elements in their 3’UTRs (32) via an undetermined pathway independent of NF-κB and MAP kinases. Future work is needed to understand the precise mechanism of how IRAK4 kinase activity mediates cytokine production. The requirement for IRAK4 kinase activity for cytokine production in human immune cells such as monocytes support the concept that IRAK4 inhibitors can be developed as potential therapeutic agents for treating autoimmune and inflammatory diseases including lupus and rheumatoid arthritis. by gest on O cber 2, 2017 hp://w w w .jb.org/ D ow nladed from IRAK4 Kinase Activation and Cytokine Induction