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RECEPTORS AND SIGNAL TRANSDUCTION
Departments of 1Biochemistry and Molecular Biology and 2Cellular and Integrative Physiology, the Walther Oncology Center, Indiana University School of Medicine and the Walther Cancer Institute, Indianapolis, Indiana
Submitted 19 October 2007 ; accepted in final form 12 June 2008
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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B is an essential regulator of the innate immune response that functions as the first line of defense against infections. Activation of the innate immune response by bacterial lipopolysaccharide (LPS) triggers production of tumor necrosis factor-
(TNF-) followed by interleukin-1 (IL-1). The IL-1 receptor associated kinase-1 (IRAK-1) is an integral component of the LPS, TNF-, and IL-1 signaling pathways that regulate NF-B. Thus we hypothesized that IRAK-1 coordinates cellular NF-B responses to LPS, TNF-, and IL-1. In contrast to TNF- where IRAK-1 subcellular localization does not change, treatment with LPS or IL-1 leads to a loss in cytoplasmic IRAK-1 with a coordinate increase in plasma membrane associated modified IRAK-1. In fibroblasts lacking the type 1 TNF- receptor (TNF R1), IRAK-1 turnover is altered and modification of IRAK-1 in the plasma membrane is decreased in response to LPS and IL-1, respectively. When NF-B controlled gene expression is measured, fibroblasts lacking TNF R1 are hyperresponsive to LPS, whereas a more variable response to IL-1 is seen. Further analysis of the LPS response revealed that plasma membrane-associated IRAK-1 is found in Toll 4, IL-1, and TNF R1-containing complexes. The data presented herein suggest a model whereby the TNF R1-IRAK-1 interaction integrates the cellular response to LPS, TNF-, and IL-1, culminating in a cell poised to activate TNF--dependent NF-B controlled gene expression. In the absence of TNF R1-dependent events, exposure to LPS or IL-1 leads to hyperactivation of the inflammatory response.
TNF-; cytokines; ubiquitinylation; cytoplasmic; phosphorylation
B leading to the transcriptional induction of over 200 genes (32). Collectively, the products of the NF-B-regulated genes are involved in the control of fundamental cellular processes such as proliferation and differentiation, as well as in the regulation of the differentiated function of cells, which includes the production of chemokines, cytokines, cell adhesion molecules, and matrix metalloproteases. Dysregulation of NF-B-dependent transcription is linked to the complications associated with chronic health disorders such as diabetes, cardiovascular diseases, autoimmune disorders and certain cancers (7, 9, 43).
In recent years significant insight has been gained regarding the signal transduction pathways that regulate NF-B activity. The majority of these signaling pathways initiate at the plasma membrane and converge at the level of the IKK complex, which contains IB kinase- (IKK), IKKβ, and the scaffold protein IKK
. The signal-dependent mechanism(s) leading to IKK complex activation remains unclear. Genes induced by NF-B regulate and mediate the innate immune response and include cytokines [tumor necrosis factor- (TNF-), interleukin-1β (IL-1)], chemokines (IL-8), immunoreceptors (MHC-I, MHC-II, IL-2R -chain), cell adhesion molecules (E-selectin; intracellular adhesion molecule-1), and acute-phase proteins (complement factor B and C4) (2, 35).
During an innate immune response, bacterially derived lipopolysaccharide (LPS) induces the release of cytokines from macrophages as well as the production of proinflammatory cytokines by surrounding cells. Intravenous LPS administration to baboons elicits a time-dependent increase in the plasma levels of TNF- (peak 1.5 h postinjection), IL-1β (peak 3 h postinjection), and IL-6 (increase after peak IL-1 level) (12, 41). If baboons are treated with neutralizing TNF- antibody prior to LPS injection, the steady-state levels of IL-1 and IL-6 do not increase. These data reveal that LPS triggers a cytokine cascade in which TNF- production followed by activation of TNF- signaling pathway(s) is required for the IL-1 and IL-6 cytokine cascade to proceed. A key cellular response that occurs after cellular stimulation with LPS, TNF-, or IL-1 is activation of NF-B-controlled gene expression. Whether the activation of NF-B by LPS, TNF-, or IL-1 is a coordinated response is unknown.
Several years ago our laboratory discovered the mouse pelle-like kinase (mPLK). The mPLK protein is the mouse homologue of the Drosophila pelle serine/threonine kinase, and a member of the serine/threonine innate immunity kinases (3, 42). Consistent with the presence of an amino terminal death domain, analysis of mPLK catalytic activity revealed that it is required for full TNF- induction of NF-B activity (8, 44). mPLK is the mouse homologue of the human interleukin-1 receptor-associated kinase-1 (IRAK-1) and hereafter is referred to as IRAK-1 (5, 42). IRAK-1 protein, but not its intrinsic kinase activity, is required for full activation of IL-1- or LPS-induced NF-B activity (29, 36). Analysis of IRAK-1 knockout mice, as well as fibroblasts derived from these mice, has revealed that IRAK-1 protein is required for LPS, TNF-, as well as IL-1 induced NF-B activity (39, 40).
Although IRAK-1 is a component of the LPS, TNF-, and IL-1 signaling pathways, the precise role of IRAK-1 in any individual pathway is not entirely clear. In response to LPS treatment, a complex containing Toll-like receptor 4 (TLR4), myeloid differentiation primary response gene 88 (MyD88), IRAK-4, and IRAK-1 forms. IRAK-1 is phosphorylated and undergoes a ubiquitin-dependent modification that targets IRAK-1 for degradation (1, 31). Current IL-1 signaling models predict that in response to IL-1, a plasma membrane-associated complex containing the type I IL-1 receptor (IL-1RI) and the IL-1RI accessory protein (IL-1RAcP) forms, IRAK-1 undergoes an IRAK-1 (and/or IRAK-4)-mediated phosphorylation, leading to the formation of a hyperphosphorylated IRAK-1 protein that is targeted within 10 min in an ubiquitin-dependent manner to the proteasome for degradation (1, 47). The sites of phosphorylation in IRAK-1 linked to IL-1 induced degradation are outside the kinase domain in a region spanning amino acids 131–144 (22, 48). When overexpressed in wild-type mouse embryo fibroblasts, catalytically inactive IRAK-1 is phosphorylated. Thus intrinsic kinase activity is not required for IRAK-1 phosphorylation. The significance of the LPS- or IL-1-induced IRAK-1 phosphorylation and ubiquitination is unclear because catalytically inactive IRAK-1 mutants do not block LPS- or IL-1-induced NF-B activity (29, 45).
Under steady-state conditions IRAK-1 can be found in TNF- type 1 receptor (TNF R1) immunocomplexes (44). In response to TNF- the level of TNF R1 associated IRAK-1 increases and IRAK-1 undergoes phosphorylation (21, 44). Results of in vitro kinase assays performed with a peptide corresponding to the IRAK-1 activation loop as a substrate suggest that IRAK-1 is capable of phosphorylating residues located in the IRAK-1 activation loop (26). These data suggest a model in which TNF- binding induces TNF R1 dimerization/oligomerization, leading to dimerization/oligomerization of associated IRAK-1 molecules, resulting in transphosphorylation and activation of IRAK-1 intrinsic kinase activity. The activated IRAK-1 kinase phosphorylates the p65 coactivator, SIMPL, leading to its nuclear accumulation (23, 28).
Why the cellular response to LPS as well as the cytokines TNF- and IL-1β, which are produced in response to LPS, depends on IRAK-1 protein for activation of NF-B controlled gene expression is unclear. To date the influence of these three stimuli on IRAK-1 protein has not been compared simultaneously. In this study the influence of LPS, TNF-, and IL-1β on IRAK-1 protein turnover and subcellular localization was examined. Our results reveal that LPS and IL-1β regulation of IRAK-1 at the plasma membrane and in the cytosol requires TNF R1 and suggest a model in which at least part of the process through which LPS initiates activation of the innate immune response is through the priming of the TNF R1 signaling pathway.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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, recombinant human IL-1β, β-actin antibody, and MG132 were purchased from Sigma (St. Louis, MO). IRAK-1, TNF R1, TLR4, Akt, lamin B, MyD88, and ubiquitin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). IL-1RAcP antibody was purchased from Chemicon (Temecula, CA). GAPDH antibody was purchased from Biodesign International (Saco, ME). EGFR and IRAK-1 antibody were purchased from Upstate (Lake Placid, NY). TNF R1-deficient fibroblasts were kindly provided by Dr. Michelle Kelliher (University of Massachusetts). Cell lysate preparation, immunocomplexing assays, and Western analysis. Media was aspirated from cell monolayers and monolayers were rinsed with ice-cold PBS or HBSS. Cells were collected with a rubber policeman in ice-cold PBS or HBSS and transferred to Eppendorf tubes. Cell pellets were collected by centrifugation (3,200 g 5'; 4°C). Cell pellets were resuspended in cytoplasmic extraction buffer [10 mM HEPES, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.2 mM PMSF, and 2.5 protease inhibitor cocktail tablets (Complete Mini Protease Inhibitor, Roche, Indianapolis, IN) per 100 ml of buffer]. Cells were incubated on ice for 25 min, cytoplasmic ("soluble") fractions were separated by centrifugation (16,000 g, 10'; 4°C), and supernates were transferred to clean Eppendorf tubes. The pelleted materials were resuspended in immunoprecipitation (IP) lysis buffer (10 mM HEPES, 150 mM or 400 mM NaCl, 5 mM EDTA, 1% Triton X-100 with 2.5 protease inhibitor cocktail tablets per 100 ml) and incubated on ice for 25 min followed by centrifugation (16,000 g, 10'; 4°C). Solubilized materials ("soluble membrane") were transferred to clean Eppendorf tubes. Soluble membrane fractions, normalized by protein concentration, were used to generate immunocomplexes with the indicated antibody. Lysates were precleared with 30 µl of protein A Sepharose beads for 1 h at 4°C, followed by an overnight incubation with the indicated antibody and protein A Sepharose beads. Immunocomplexes were collected by centrifugation (8,200 g, 10 s, 25°C). Pelleted material was resuspended in IP wash buffer (10 mM HEPES, 150 mM or 400 mM NaCl, 5 mM EDTA, 0.1% Triton X-100, with 2.5 mini protease inhibitor cocktail tablets per 100 ml) and centrifuged, and the process was repeated for a total of three washes. In experiments employing only Western analysis, soluble and soluble membrane fractions were generated and normalized by protein concentration. All samples were denatured by boiling for 5 min in Laemmli buffer containing β-mercaptoethanol and subjected to SDS-PAGE as previously described (44). Stripping buffer (100 mM β-mercaptoethanol, 62 mM Tris·HCl, pH 6.7, 2% SDS, double distilled water) was used to remove antibody from blots were indicated. Blots were incubated in 50°C stripping buffer for 15 min with agitation followed by 3–5 min washes and Western analysis (44).
Differential ultracentrifugation.
Approximately 1 x 107 MEFs were treated with LPS prior to subcellular fractionation. MEFs were fractionated by differential ultracentrifugation as described by Elmendorf (11) with the following exceptions. Cells were quickly washed two times in ice-cold HES buffer (20 mM HEPES, 1 mM EDTA, and 255 mM sucrose, pH 7.4, 2 mM PMSF, 1 complete protease tablet per 25 ml of buffer), flash frozen in liquid nitrogen and stored at –80°C. Prior to lysis, cells were slightly thawed on ice in 2 ml of fresh ice-cold HES buffer, scraped, and transferred to an Oakridge tube. MEFs were lysed by 10 passes through a 23
-gauge needle (Becton Dickinson, Franklin Lakes, NJ). The homogenate was centrifuged at 19,000 g for 20 min at 4°C. The supernatant [cytosol, endoplasmic reticulum (ER), golgi] was removed and centrifuged at 180,000 g for 75 min at 4°C to separate the ER and Golgi from the cytosol. The pellet from the first spin was resuspended in 5 ml of HES buffer, vortexed, layered onto a 6.3 ml sucrose cushion (1.12 M), and centrifuged at 100,000 g for 60 min at 4°C which produced two fractions, plasma membranes (interface) and nuclei (pellet). The interface (plasma membrane) was removed, resuspended in 10 ml of HES buffer, repelleted at 27,000 g at 4°C for 30 min and then resuspended in HES buffer. The nuclear pellet was also resuspended in HES buffer. Equal amounts of protein from the plasma membrane, cytosol, and nucleus were separated by 10% SDS-PAGE.
EMSAs.
EMSAs were performed as described previously (15). Nuclear extracts were isolated from mouse embryo fibroblasts (wild-type or IRAK-1–/Y) as described by Schreiber et al. (34). Oligonucleotides containing an NF-B binding site (Promega, Madison, WI) were end labeled with [-32P]ATP and T4 polynucleotide kinase according to supplier specifications. EMSAs contained (in 20 µl) 1 µg of nuclear extract, 3 fmol of synthetic double-stranded oligonucleotide (upper strand: 5'-AGT TGA GGG GAC TTT CCC AGG C.-3'), and 50 ng of poly(dI-dC) in binding buffer (50 mM Tris, pH 7.9, 1 mM EDTA, 1 mM dithiothreitol, 12.5 mM MgCl2, 10% glycerol, and 0.1 M KCl). After incubation for 15 min on ice, assays were loaded onto 4% acrylamide gels (39:1, acrylamide-bisacrylamide) containing 5% glycerol that had been prerun for 30 min in a 0.25x TBE buffer (1x = 0.89 M Tris borate, 0.089 M boric), dried, and exposed to Kodak XAR-5 film overnight.
Real-time PCR.
Total RNA was isolated from MEFs using the RNeasy Plus Mini Kit (Qiagen, Valencia, CA). The High Capacity Reverse Transcriptase Kit (Applied Biosystems, University Park, IL) was used to make cDNA from 2 µg of total RNA. The reaction volume for real-time PCR was 25 µl [12.5 µl SYBR Green Master Mix (Applied Biosystems), 1 µl forward primer (final concentration is 0.4 µM), 1 µl reverse primer (final concentration is 0.4 µM), 5 µl cDNA (50 ng), 5.5 µl PCR grade water (Ambion, Austin, TX)]. The sequences are as follows: IkB forward 5'-CTG CAG GCC ACC AAC TAC AA-3', IkB reverse 5'-CAG CAC CCA AAG TCA CCA AGT-3'; MCP-1 forward 5'-CCA CTC ACC TGC TGC TAC TCA T-3', MCP-1 reverse 5'-TGG TGA TCC TCT TGT AGC TCT CC-3'; β-actin forward 5'-AGG TGT GCA CCT TTT ATT GGT CTC AA-3', β-actin reverse 5'-TCT ATG AAG GTT TGG TCT CCC T-3'; TNF- forward 5'-TGT CTC AGC CTC TTC TCA TT-3', TNF- reverse 5'-TGA TCT GAG TGT GAG GGT CT-3'; IL-6 forward 5'-TGT GCA ATG GCA ATT CTG AT-3', IL-6 reverse 5'-GGT ACT CCA GAA GAC CAG AGG A-3'. PCR parameters are as follows: stage 1, 50°C for 2 min; stage 2, 95°C for 10 min; stage 3, step 1 = 95°C for 15 s for 45 cycles, step 2 = 60°C for 1 min (data collection); stage 3, step 1 = 95°C for 15 s, step 2 = 60°C for 15 s, step 3 = 95°C for 15 s; (dissociation curve) stage 4, –72°C hold. Primer sequences were a kind gift from Dr. Brian Ashburner (University of Toledo, Toledo, OH) and were purchased from IDCT (Coralville, IA).
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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, and IL-1 NF-B activation.
To compare the requirement for IRAK-1 in LPS-, TNF--, or IL-1-induced activation of NF-B, we examined, simultaneously, the effect of each stimulus on NF-B DNA binding activity in MEFs derived from wild-type or IRAK-1-deficient mice (IRAK-1–/Y). NF-B DNA binding activity was diminished in MEFs derived from the IRAK-1–/Y compared with MEFs derived from wild-type animals following LPS, TNF-, or IL-1 treatment (Fig. 1). These data are similar to studies published by others for TNF- and IL-1 and confirm a role for IRAK-1 in LPS-induced NF-B DNA binding activity (39). In these experiments, on a molar basis, TNF- is the most potent inducer of NF-B DNA binding activity (0.2 nM compared with 0.5 nM and 1–2 nM for IL-1 and LPS, respectively). Thus induction of NF-B DNA binding activity by LPS as well as the cytokine cascade induced by LPS are linked by a requirement for IRAK-1.
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induction of NF-B-inducible genes is attenuated in IRAK-1-deficient MEFs.
The link between IRAK-1 and IL-1 or LPS-induced NF-B activity is widely appreciated, whereas the link between TNF- and IRAK-1 is less well recognized. Therefore we determined whether the reduced p65 binding in TNF--treated IRAK-1–/Y fibroblasts reflected a decrease in TNF- induction of NF-B-dependent genes. Compared with wild-type fibroblasts, there is a 75% reduction in IB and MCP-1 gene expression in TNF--treated IRAK-1–/Y fibroblasts (Fig. 2). These data confirm that IRAK-1 is a component of the signaling pathway(s) through which TNF- induces activation of NF-B-controlled genes. In combination with the work of others (39), the data presented in Figs. 1 and 2 reveal that induction of NF-B DNA binding activity by LPS as well as the cytokine cascade induced by LPS are linked by a requirement for IRAK-1.
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, and IL-1.
Protein phosphorylation can have a profound effect on protein activity and stability as well as subcellular localization. IRAK-1 is posttranslationally modified (phosphorylation, ubiquitinylation) in cells treated with either LPS or IL-1, which leads to a slower migrating form of IRAK-1 in SDS-PAGE (27, 47). To compare the kinetics of the IRAK-1 posttranslational modifications, MEFs were treated with LPS, TNF-, or IL-1 for varying periods of time. Extracts from treated cells were separated into cytoplasmic and soluble membrane fractions, which were subjected to Western analysis. In response to either LPS or IL-1, but not TNF-, there is a time-dependent decrease in the cytoplasmic pool of unmodified IRAK-1 coordinated with a time-dependent increase in modified IRAK-1 in the soluble membrane fraction (Fig. 3A). In contrast to LPS and IL-1, TNF- treatment did not lead to a change in the apparent molecular mass or abundance of the IRAK-1 protein in either the cytoplasmic or the soluble membrane fractions (Fig. 3B; the increased signal in the 1- and 4-h time points is due to background binding). These data are consistent with previous studies demonstrating that TNF- treatment of fibroblasts (5 min) leads to an increase in [32P]ATP incorporation into IRAK-1 without a change in the apparent molecular mass (21).
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The buffer used to generate the soluble subcellular fractions lacks detergent and crudely separates cells into fractions enriched in cytoplasmic and membrane/nuclear components (soluble membrane). To confirm that the subcellular localization of the modified version of IRAK-1 was in fact the plasma membrane, differential centrifugation was used to generate plasma membrane, cytosolic, nuclear, and ER/Golgi-enriched fractions (11, 33). In the absence of stimulation, unmodified IRAK-1 is found in all fractions (Fig. 3B). In response to LPS modified IRAK-1 can be detected primarily in the plasma membrane and ER/Golgi-enriched fractions (data not shown), with trace amounts in the nuclear fraction (Fig. 3B).
LPS-induced IRAK-1 modification(s) is proteasome sensitive.
IRAK-1 undergoes a rapid phosphorylation and ubiquitin-dependent degradation in IL-1-treated cells (47). In response to LPS treatment, the cellular pool of IRAK-1 is also predicted to undergo phosphorylation as well as ubiquitinylation. The steady-state level of IRAK-1 protein is reduced in the cytosolic and soluble membrane fractions 4 h after fibroblasts are treated with LPS (Fig. 3A). In IL-1-treated cultures, ubiquitinylation leads to proteasomal-dependent degradation of IRAK-1 (47). IRAK-1 is thought to undergo ubiquitin-dependent degradation in response to LPS (16). Since phosphorylation often targets proteins for ubiquitin-dependent degradation, we examined whether LPS targets IRAK-1 for proteasomal degradation. In cultures pretreated with the proteasome inhibitor MG132, modified IRAK-1 accumulates in the soluble membrane fraction in LPS-treated cultures, which demonstrates that IRAK-1 degradation is proteasome dependent (Fig. 4A). Western analysis of IRAK-1 immunocomplexes with ubiquitin antibody confirm the proposition that IRAK-1 is ubiquitinylated in response to LPS (Fig. 4B). To determine whether IRAK-1 is also subject to degradation by the lysosome, a subcellular organelle that also mediates protein turnover, the effect of chloroquine on the stability of modified IRAK-1 was examined. Pretreatment with chloroquine had no effect on the cytoplasmic or soluble membrane fractions of IRAK-1 in LPS-treated cultures (Fig. 4C). In addition, the poly-ADP ribosylation inhibitor 3-aminobenzomide has been shown to block NF-B transactivation, but it has no effect on IRAK-1 modification and degradation (Fig. 4C). Thus LPS and IL-1 both target cytosolic IRAK-1 to the plasma membrane, and LPS, like IL-1, induces ubiquitin-dependent degradation of IRAK-1 by the proteasome. However, a key difference between LPS and IL-1 is in the kinetics of the response: IL-1 elicits a rapid response (within 30 min) whereas the response to LPS is delayed (60 min).
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plasma levels peaks between 1 and 1.5 h, whereas the IL-1 plasma levels does not peak until 3 h (12); we therefore focused on the kinetics of the LPS-induced TNF R1-IRAK-1 complex formation. In parallel to the peak blood levels of TNF-, LPS-induced TNF R1-IRAK-1 complex formation peaks within an hour of LPS treatment (Fig. 6A). Four hours after LPS treatment, TNF R1-IRAK-1 complexes are absent; however, they are still present in cells treated with the proteasome inhibitor MG132 (Fig. 6B).
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receptor and the IL-1 receptor accessory protein. These data led us to test the functional significance of the LPS-induced TNF R1-IRAK-1 and IL-1RAcP-IRAK-1 interactions. The first step in the proinflammatory cascade induced by LPS is TNF- production. Thus we examined whether the absence of TNF R1-IRAK-1 complex formation would alter the effect of LPS on IRAK-1 protein. Wild-type fibroblasts and fibroblasts derived from TNF R1–/– mice were treated with LPS for the indicated times followed by separation into soluble membrane and cytosolic fractions. Western analysis revealed that LPS-induced IRAK-1 modification is slightly reduced in soluble membranes isolated from TNF R1–/– fibroblasts compared with wild-type fibroblasts (Fig. 7A). Interestingly, unmodified IRAK-1 is detectable for a longer period of time in both the cytosol and solubilized membranes of TNF R1–/– fibroblasts compared with wild-type fibroblasts. Analysis of other TLR4 signaling pathway components revealed that MyD88 is only associated with the soluble membrane fraction. In wild-type fibroblasts there is a slight increase in membrane associated MyD88 1 h after LPS treatment followed by a return to the prestimulated level by 4 h (Fig. 7A). In contrast, the MyD88 levels appear to increase throughout the course of LPS treatment in TNF R1–/– fibroblasts. IRAK-4 protein levels differ when the wild-type and TNF R1–/– fibroblasts are compared. In wild-type fibroblasts IRAK-4 steady-state levels decline, whereas in the absence of TNF R1, IRAK-4 steady-state levels appear to increase in response to LPS.
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B-dependent gene expression would be affected in cells lacking the ability to generate TNF R1-IRAK-1 complexes. Wild-type and TNF R1–/–-derived fibroblasts were treated with LPS for the indicated time periods and the abundance of IB, TNF-, and IL-6 mRNAs were measured by quantitative real-time PCR (Fig. 7B). In contrast to IB where the absence of TNF R1-IRAK-1 complex formation had no effect, TNF- mRNA levels were dramatically increased in LPS-treated TNF R1–/– MEFs (13- vs. 156-fold). TNF- message returned to basal levels in wild-type and TNF R1–/– fibroblasts at the latest LPS time point tested (24 h). Similarly, IL-6 gene expression is dramatically affected in the LPS-treated TNF R1–/– fibroblasts. In LPS-treated wild-type fibroblasts IL-6 transcripts are increased by 26- and 22-fold at 4 and 24 h, respectively, whereas in the LPS-treated TNF R1–/– fibroblasts at the same time points the IL-6 transcript levels are increased, 81- and 83-fold, respectively.
TNF R1 affects IL-1 induced NF-B activity.
As demonstrated directly above, LPS signaling is coordinated in a manner that involves TNF R1 and IRAK-1. Therefore, it was of interest to determine whether IL-1 responsiveness would be affected in cells lacking the ability to generate TNF R1-IRAK-1 complexes. Wild-type and TNF R1–/–-derived fibroblasts treated with IL-1 for the indicated times were separated into soluble membrane and cytosolic fractions. As was done for the LPS-treated fibroblasts, the steady-state levels of IRAK-1, MyD88, and IRAK-4 proteins were examined by Western analysis. Although modified IRAK-1 is detected in the soluble membrane fraction in wild-type and TNF R1-deficient fibroblasts, there is noticeably less modified IRAK-1 in the IL-1-treated TNF R1-deficient fibroblasts (Fig. 8A). In contrast to the LPS response, the rate of decline in the cytosolic pool of IRAK-1 does not appear to differ in IL-1-treated wild-type vs. TNF R1-deficient fibroblasts. Also in contrast to the LPS response, steady-state levels of MyD88 change in a parallel fashion in IL-1-treated wild-type and TNF R1-deficient fibroblasts (Fig. 8A). Again in contrast to the LPS response, IRAK-4 regulation is considerably different in IL-1-treated TNF R1–/– fibroblasts. Ten minutes after IL-1 treatment IRAK-4 levels start to decline in TNF R1–/– fibroblasts, whereas they remain relatively steady in IL-1-treated wild-type fibroblasts.
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B-dependent gene expression was also examined. In contrast to LPS, however, during early time points following IL-1 treatment, IB expression is considerably reduced in the absence of TNF R1 (16-fold vs. 30-fold in wild-type MEFs). IB message returns to wild-type levels in the TNF R1–/– fibroblasts at later IL-1 time points (4 and 24 h). Under basal conditions TNF- is not expressed in wild-type or TNF R1–/– MEFs (Figs. 7B and 8B). However, in the absence of TNF R1, TNF- gene expression is detectable earlier (0.5 h) in IL-1-treated TNF R1-deficient fibroblasts compared with wild-type fibroblasts. One hour after IL-1 treatment TNF- gene expression is enhanced in TNF R1–/– compared with wild-type fibroblasts (64-fold vs. 50-fold, respectively). Furthermore, TNF R1-deficient fibroblasts express TNF- for a longer period of time in response to IL-1, sevenfold vs. basal levels in wild-type fibroblasts at 4 h. IL-1 elicits an increase in IL-6 gene expression in wild-type and TNF R1–/– fibroblasts. However, in the TNF R1-deficient fibroblasts IL-6 expression is dramatically enhanced at later time points (95-fold vs. 27-fold in wild-type at 4 h and 173-fold vs. 35-fold in wild-type at 24 h). |
DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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B-regulated genes. Analysis of cytokine and cytokine receptor knockout animals has revealed that different cytokines mediate different components of the host response (14). However, the mechanism through which the host response integrates the individual cytokines is not clear. In vitro LPS is used as a surrogate inducer to examine cellular responses to gram-negative bacteria. LPS elicits a time-dependent increase in circulating levels of TNF- (peak 1 h) and IL-1 (peak 3 h). Since IRAK-1 is linked to the induction of NF-B activity by LPS, TNF-, and IL-1 (36, 39, 44), we set out to determine whether IRAK-1 integrates cellular responses to LPS, TNF-, and IL-1.
Current models predict that engagement of TLR-4 by LPS or IL-1R1 by IL-1 leads to formation of a protein complex at the receptor. MyD88 and MyD88 adapter-like (Mal) in LPS signaling is recruited to the receptor leading to recruitment of IRAK-4 and IRAK-1 via death domain interactions (46). IRAK-4 phosphorylates IRAK-1 leading to IRAK-1 activation (22). Catalytically active IRAK-1 autophosphorylates, dissociates from the receptor, and forms a complex at the plasma membrane that contains the ubiquitin ligase TRAF-6, TAB2, which binds lysine 63-linked ubiquitin chains, and the protein kinase TAK-1 (25). Membrane-associated IRAK-1 is ubiquitinylated and eventually degraded by the proteasome (20, 47). The TRAF-6, TAK-1, and TAB1/2 complex phosphorylates and activates the IKK complex, resulting in the induction of NF-B-regulated gene expression (19).
Results of our studies reveal that the IRAK-1 protein is regulated in two distinct manners by the LPS and IL-1 signaling pathways. LPS or IL-1 treatment leads to a time-dependent loss of cytoplasmic pool of IRAK-1 that occurs rapidly in response to IL-1 (within 10 min) and more slowly in response to LPS (60 min) (Fig. 3). The IL-1 data support those of Yamin and Miller (47), who demonstrated that within 5 min of IL-1 treatment IRAK-1 is phosphorylated and targeted for ubiquitin-dependent degradation by the proteasome. In a parallel manner, LPS also induces ubiquitin-dependent degradation of IRAK-1 protein but with slower kinetics (Figs. 3A and 4). Inhibition of lysosomal activity has no effect on the steady-state level of IRAK-1 protein; thus we conclude that IRAK-1 protein turnover is mediated by the proteasome (Fig. 4). Based on the data presented herein and the work of others (50), a model can be postulated in which the cytoplasmic pool of IRAK-1 is targeted to the plasma membrane, where it undergoes posttranslational modification (phosphorylation and ubiquitinylation) that eventually leads to its degradation. However, the kinetics of the LPS and IL-1 responses indicate that the membrane-targeted IRAK-1 is turned over much more rapidly following IL-1 compared with LPS treatment.
In response to LPS, modified IRAK-1 is found primarily in plasma membrane and ER/Golgi. Unmodified IRAK-1 was detected in the nuclear fraction, an observation that has been made by others (4, 18). A limitation of the subcellular fractionation approach used by ourselves and Huang et al. (18), however, is that preparing nuclear fractions free from contaminating ER/Golgi is challenging, since the outer membrane of the nuclear envelop is contiguous with the ER. Although Bol et al. (4) used immunofluorescence to detect nuclear IRAK-1, only the results of a single cell were shown. However, we cannot rule out the possibility that LPS targets IRAK-1 to the nucleus. Currently the role of nuclear IRAK-1 is unclear.
In TNF R1–/– fibroblasts, the LPS and IL-1 effects on IRAK-1 protein are altered, which further strengthens our hypothesis that IRAK-1 is an integral component of the three signaling pathways. In LPS-treated TNF R1-deficient fibroblasts, turnover of the cytoplasmic pool of IRAK-1 is delayed; modified IRAK-1 is detected in the soluble membrane fraction; however, its turnover at the plasma membrane through protein degradation and/or trafficking is also delayed (Fig. 7A). In IL-1-treated TNF R1–/– fibroblasts, the steady-state levels of cytoplasmic and plasma membrane IRAK-1 are unaffected. Instead the amount of modified IRAK-1 in the plasma membrane is decreased (Fig. 8A). Taken together our results demonstrate that either IRAK-1 ubiquitinylation occurs at the plasma membrane or ubiquitinylation of IRAK-1 targets it for the plasma membrane. TNF R1 is not required for the ubiquitinylation of IRAK-1, but either trafficking or retention of IRAK-1 protein in the plasma membrane occurs less efficiently in the absence of TNF R1.
Analysis of other LPS and IL-1 signaling pathways components revealed that MyD88, like IRAK-1, is expressed at equivalent levels under steady-state conditions in the wild-type and TNF R1-deficient fibroblasts (Figs. 7 and 8). In contrast, the steady-state level of IRAK-4 is lower in the TNF R1-deficient fibroblasts. IRAK-4 is required for NF-B activation by LPS and IL-1 (35a). Therefore, the difference in IRAK-4 levels may explain some of the differences in expression of NF-B controlled genes in the LPS and IL-1-treated TNF R1-deficient fibroblasts: the dramatic increase in TNF- and IL-6 mRNA in LPS-treated TNF R1–/– fibroblasts and the dramatic increase in IL-6 but not TNF- in IL-1-treated TNF R1-deficient fibroblasts (Figs. 7 and 8). Although TNF- IB and IL-6 are NF-B-inducible genes, our studies do not discriminate between the contributions of NF-B and other transcription factors, such as AP1 and C/EBPβ, which are also activated by LPS and IL-1. Takada and Aggarwal (37) reported enhanced NF-B DNA binding in LPS-treated TNF R1–/– macrophages, increased activation of c-Jun terminal kinase, and upregulation of COX-2 and NO synthase proteins. These data lead the authors to suggest cross talk between the LPS and TNF- signaling pathways. On the basis of the data presented herein, we propose that IRAK-1 is only one potential mediator of cross talk.
Independent of LPS stimulation, TNF- induces the formation of complexes between endogenous TNF R1 and endogenous IRAK-1 (44). Yet it is widely accepted that the IRAK-1 protein, independent of its kinase activity, has a scaffolding function in the LPS and IL-1 signaling pathways. A growing body of evidence suggests that IRAK-1 kinase activity is a critical component of the TNF R1 pathway (28, 44). Analysis of fibroblasts derived from IRAK-1–/Y animals has confirmed the requirement for IRAK-1 in TNF- induced NF-B activity (39, 44). A striking example of IRAK-1 involvement in the TNF- signaling pathway is observed in IRAK-1 null/TNF- transgenic mice. Transgenic mice that overexpress TNF- in the heart develop diminished cardiac contractility and die of heart failure within 5 mo of birth (40). When the TNF- transgene is crossed into an IRAK-1–/Y background, 70% of the mice are alive at 5 mo. These data parallel the human situation in which heart failure can be associated with elevated circulating levels of TNF- (6). In a phase I study conducted with Enbrel, treatment with this TNF- antagonist improved myocardial function in heart failure patients (10). Thus at both biochemical and genetic levels IRAK-1 activity is linked to TNF- function.
The function of IRAK-1 catalytic activity in the TNF- pathway is phosphorylation of SIMPL, a p65-specific coactivator (23, 28). NF-B translocates to the nucleus in response to TNF-. However, IRAK-1 phosphorylation of SIMPL and its subsequent nuclear localization is required for full activation of NF-B-inducible genes (23, 28). Herein, we present genetic evidence that IRAK-1 is an important mediator of TNF- signal transduction as IRAK-1–/Y fibroblasts have reduced expression of two NF-B-inducible genes, IB and MCP-1 (decreased 75%) in response to TNF- (Fig. 2). Since neither inhibition of SIMPL nuclear localization nor deletion of IRAK-1 completely attenuates TNF- induced NF-B transactivation, it is possible that IRAK-1 and SIMPL are jointly responsible for activation of a specific subset of NF-B-controlled genes.
Our studies have identified IRAK-1 as a molecule that integrates the LPS, TNF-, and IL-1 signaling pathways. LPS targets IRAK-1 to TNF R1 and IL-1RAcP (Fig. 5). These data argue that LPS primes cells to respond to TNF- and IL-1 by mobilizing a common signaling pathway component, IRAK-1, to bind to TNF R1 and IL-1RAcP. The concept of coordinate signaling among LPS, TNF-, and IL-1β-activated pathways is not new. Han et al. (13) discovered that LPS induces biphasic activation of NF-B and that the second phase results from activation of TNF- and IL-1β signaling pathways. Furthermore, pretreatment of cells or animals with LPS or TNF- prior to acute LPS or TNF- treatment has a profound affect on signaling. Pretreatment with LPS or TNF- leads to hyporesponsiveness or tolerance to subsequent LPS stimulation by reducing NF-B DNA binding (24, 38). Laegreid et al. (24) speculate that pretreatment with TNF- either depletes or inactivates signaling proteins that are involved in LPS signaling events. Furthermore, analysis of mice lacking TNF- reveals that not only is activation of the inflammatory response compromised but the ability to turn off an activated response is also compromised (28a). Our data demonstrating that TNF R1–/– MEFs are hyperresponsive to LPS and IL-1 as evidenced by the dramatic increase TNF- and IL-6 mRNA expression are consistent with the TNF- knockout phenotype. Thus transactivation of NF-B inducible genes is extremely distorted in TNF R1-deficient fibroblasts treated with LPS or IL-1. We propose that TNF R1 mediates a regulatory signaling loop that controls the duration and intensity of the inflammatory response. Although regulated integration of the LPS, TNF-, and IL-1β pathways has been proposed previously our data suggest that IRAK-1 mediates integration of these pathways.
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2. Baeuerle PA, Henkel T. Function and activation of NF-kappa B in the immune system. Annu Rev Immunol 12: 141–179, 1994.[Web of Science][Medline]
3. Belvin MP, Anderson KV. A conserved signaling pathway: the Drosophila toll-dorsal pathway. Annu Rev Cell Dev Biol 12: 393–416, 1996.[CrossRef][Web of Science][Medline]
4. Bol G, Kreuzer OJ, Brigelius-Flohe R. Translocation of the interleukin-1 receptor-associated kinase-1 (IRAK-1) into the nucleus. FEBS Lett 477: 73–78, 2000.[CrossRef][Web of Science][Medline]
5. Cao Z, Henzel WJ, Gao X. IRAK: a kinase associated with the interleukin-1 receptor. Science 271: 1128–1131, 1996.[Abstract]
6. Ceconi C, Curello S, Bachetti T, Corti A, Ferrari R. Tumor necrosis factor in congestive heart failure: a mechanism of disease for the new millennium? Prog Cardiovasc Dis 41: 25–30, 1998.[CrossRef][Web of Science][Medline]
7. Chen F. Is NF-kappaB a culprit in type 2 diabetes? Biochem Biophys Res Commun 332: 1–3, 2005.[CrossRef][Web of Science][Medline]
8. Cooke EL, Uings IJ, Xia CL, Woo P, Ray KP. Functional analysis of the interleukin-1-receptor-associated kinase (IRAK-1) in interleukin-1 beta-stimulated nuclear factor kappa B (NF-kappa B) pathway activation: IRAK-1 associates with the NF-kappa B essential modulator (NEMO) upon receptor stimulation. Biochem J 359: 403–410, 2001.[CrossRef][Web of Science][Medline]
9. Coussens LM, Werb Z. Inflammation and cancer. Nature 420: 860–867, 2002.[CrossRef][Web of Science][Medline]
10. Deswal A, Bozkurt B, Seta Y, Parilti-Eiswirth S, Hayes FA, Blosch C, Mann DL. Safety and efficacy of a soluble P75 tumor necrosis factor receptor (Enbrel, etanercept) in patients with advanced heart failure. Circulation 99: 3224–3226, 1999.
11. Elmendorf JS. Fractionation analysis of the subcellular distribution of GLUT-4 in 3T3–L1 adipocytes. Methods Mol Med 83: 105–111, 2003.[Medline]
12. Fong Y, Tracey KJ, Moldawer LL, Hesse DG, Manogue KB, Kenney JS, Lee AT, Kuo GC, Allison AC, Lowry SF. Antibodies to cachectin/tumor necrosis factor reduce interleukin 1 beta and interleukin 6 appearance during lethal bacteremia. J Exp Med 170: 1627–1633, 1989.
13. Han SJ, Ko HM, Choi JH, Seo KH, Lee HS, Choi EK, Choi IW, Lee HK, Im SY. Molecular mechanisms for lipopolysaccharide-induced biphasic activation of nuclear factor-kappa B (NF-kappa B). J Biol Chem 277: 44715–44721, 2002.
14. Hanada T, Yoshimura A. Regulation of cytokine signaling and inflammation. Cytokine Growth Factor Rev 13: 413–421, 2002.[CrossRef][Web of Science][Medline]
15. Harrington MA, Konicek B, Song A, Xia XL, Fredericks WJ, Rauscher FJ 3rd. Inhibition of colony-stimulating factor-1 promoter activity by the product of the Wilms' tumor locus. J Biol Chem 268: 21271–21275, 1993.
16. Hu J, Jacinto R, McCall C, Li L. Regulation of IL-1 receptor associated kinases by lipopolysaccharide. J Immunol 3910–3914, 2002.
17. Huang J, Gao X, Li S, Cao Z. Recruitment of IRAK to the interleukin 1 receptor complex requires interleukin 1 receptor accessory protein. Proc Natl Acad Sci USA 94: 12829–12832, 1997.
18. Huang Y, Li T, Sane DC, Li L. IRAK1 serves as a novel regulator essential for lipopolysaccharide-induced interleukin-10 gene expression. J Biol Chem 279: 51697–51703, 2004.
19. Jiang Z, Johnson HJ, Nie H, Qin J, Bird TA, Li X. Pellino 1 is required for interleukin-1 (IL-1)-mediated signaling through its interaction with the IL-1 receptor-associated kinase 4 (IRAK4)-IRAK-tumor necrosis factor receptor-associated factor 6 (TRAF6) complex. J Biol Chem 278: 10952–10956, 2003.
20. Jiang Z, Ninomiya-Tsuji J, Qian Y, Matsumoto K, Li X. Interleukin-1 (IL-1) receptor-associated kinase-dependent IL-1-induced signaling complexes phosphorylate TAK1 and TAB2 at the plasma membrane and activate TAK1 in the cytosol. Mol Cell Biol 22: 7158–7167, 2002.
21. Kim JA, Yeh DC, Ver M, Li Y, Carranza A, Conrads TP, Veenstra TD, Harrington MA, Quon MJ. Phosphorylation of Ser24 in the pleckstrin homology domain of insulin receptor substrate-1 by Mouse Pelle-like kinase/interleukin-1 receptor-associated kinase: cross-talk between inflammatory signaling and insulin signaling that may contribute to insulin resistance. J Biol Chem 280: 23173–23183, 2005.
22. Kollewe C, Mackensen AC, Neumann D, Knop J, Cao P, Li S, Wesche H, Martin MU. Sequential autophosphorylation steps in the interleukin-1 receptor-associated kinase-1 regulate its availability as an adapter in interleukin-1 signaling. J Biol Chem 279: 5227–5236, 2004.
23. Kwon HJ, Breese EH, Vig-Varga E, Luo Y, Lee Y, Goebl MG, Harrington MA. Tumor necrosis factor alpha induction of NF-kappaB requires the novel coactivator SIMPL. Mol Cell Biol 24: 9317–9326, 2004.
24. Laegreid A, Thommesen L, Jahr TG, Sundan A, Espevik T. Tumor necrosis factor induces lipopolysaccharide tolerance in a human adenocarcinoma cell line mainly through the TNF p55 receptor. J Biol Chem 270: 25418–25425, 1995.
25. Lamothe B, Besse A, Campos AD, Webster WK, Wu H, Darnay BG. Site-specific Lys-63-linked tumor necrosis factor receptor-associated factor 6 auto-ubiquitination is a critical determinant of I kappa B kinase activation. J Biol Chem 282: 4102–4112, 2007.
26. Li S, Strelow A, Fontana EJ, Wesche H. IRAK-4: a novel member of the IRAK family with the properties of an IRAK-kinase. Proc Natl Acad Sci USA 99: 5567–5572, 2002.
27. Li X, Commane M, Burns C, Vithalani K, Cao Z, Stark GR. Mutant cells that do not respond to interleukin-1 (IL-1) reveal a novel role for IL-1 receptor-associated kinase. Mol Cell Biol 4643–4652, 1999.
28. Luo Y, Kwon HJ, Montano S, Georgiadis M, Goebl MG, Harrington MA. Phosphorylation of SIMPL modulates RelA-associated NF-B-dependent transcription. Am J Physiol Cell Physiol 292: C1013–C1023, 2007.
28a. Marino MW, Dunn A, Grail D, Inglese M, Noguchi Y, Richards E, Jungbluth A, Wada H, Moore M, Williamson B, Basu S, Old LJ. Characterization of tumor necrosis factor-deficient mice. Proc Natl Acad Sci USA 94: 8093–8098, 1997.
29. Maschera B, Ray K, Burns K, Volpe F. Overexpression of an enzymically inactive interleukin-1-receptor-associated kinase activates nuclear factor-kappaB. Biochem J 339: 227–231, 1999.[CrossRef][Web of Science][Medline]
30. Medvedev AE, Piao W, Shoenfelt J, Rhee SH, Chen H, Basu S, Wahl LM, Fenton MJ, Vogel SN. Role of TLR4 tyrosine phosphorylation in signal transduction and endotoxin tolerance. J Biol Chem 282: 16042–16053, 2007.
31. O'Neill L. The Toll/interleukin-1 receptor domain: a molecular switch for inflammation and host defence. Biochem Soc Trans 28: 557–563, 2000.[Web of Science][Medline]
32. Pahl HL. Activators and target genes of Rel/NF-kappaB transcription factors. Oncogene 18: 6853–6866, 1999.[CrossRef][Web of Science][Medline]
33. Piper RC, Hess LJ, James DE. Differential sorting of two glucose transporters expressed in insulin-sensitive cells. Am J Physiol Cell Physiol 260: C570–C580, 1991.
34. Schreiber E, Matthias P, Muller MM, Schaffner W. Rapid detection of octamer binding proteins with ‘mini-extracts’, prepared from a small number of cells. Nucleic Acids Res 17: 6419, 1989.
35. Siebenlist U, Franzoso G, Brown K. Structure, regulation and function of NF-kappa B. Annu Rev Cell Biol 10: 405–455, 1994.[CrossRef][Web of Science][Medline]
35a. Suzuki N, Suzuki S, Duncan GS, Millar DG, Wada T, Mirtsos C, Takada H, Wakeham A, Itie A, Li S, Penninger JM, Wesche H, Ohashi PS, Mak TW, Yeh WC. Severe impairment of interleukin-1 and Toll-like receptor signalling in mice lacking IRAK-4. Nature 416: 750–756, 2002.[CrossRef][Web of Science][Medline]
36. Swantek JL, Tsen MF, Cobb MH, Thomas JA. IL-1 receptor-associated kinase modulates host responsiveness to endotoxin. J Immunol 164: 4301–4306, 2000.
37. Takada Y, Aggarwal BB. Genetic deletion of the tumor necrosis factor receptor p60 or p80 sensitizes macrophages to lipopolysaccharide-induced nuclear factor-kappa B, mitogen-activated protein kinases, and apoptosis. J Biol Chem 278: 23390–23397, 2003.
38. Tamandl D, Bahrami M, Wessner B, Weigel G, Ploder M, Furst W, Roth E, Boltz-Nitulescu G, Spittler A. Modulation of toll-like receptor 4 expression on human monocytes by tumor necrosis factor and interleukin-6: tumor necrosis factor evokes lipopolysaccharide hyporesponsiveness, whereas interleukin-6 enhances lipopolysaccharide activity. Shock 20: 224–229, 2003.[CrossRef][Web of Science][Medline]
39. Thomas JA, Allen JL, Tsen M, Dubnicoff T, Danao J, Liao XC, Cao Z, Wasserman SA. Impaired cytokine signaling in mice lacking the IL-1 receptor-associated kinase. J Immunol 163: 978–984, 1999.
40. Thomas JA, Haudek SB, Koroglu T, Tsen MF, Bryant DD, White DJ, Kusewitt DF, Horton JW, Giroir BP. IRAK1 deletion disrupts cardiac Toll/IL-1 signaling and protects against contractile dysfunction. Am J Physiol Heart Circ Physiol 285: H597–H606, 2003.
41. Tracey KJ, Fong Y, Hesse DG, Manogue KR, Lee AT, Kuo GC, Lowry SF, Cerami A. Anti-cachectin/TNF monoclonal antibodies prevent septic shock during lethal bacteraemia. Nature 330: 662–664, 1987.[CrossRef][Web of Science][Medline]
42. Trofimova M, Sprenkle AB, Green M, Sturgill TW, Goebl MG, Harrington MA. Developmental and tissue-specific expression of mouse pelle-like protein kinase. J Biol Chem 271: 17609–17612, 1996.
43. Valen G, Yan ZQ, Hansson GK. Nuclear factor kappa-B and the heart. J Am Coll Cardiol 38: 307–314, 2001.
44. Vig E, Green M, Liu Y, Donner DB, Mukaida N, Goebl MG, Harrington MA. Modulation of tumor necrosis factor and interleukin-1-dependent NF-kappaB activity by mPLK/IRAK. J Biol Chem 274: 13077–13084, 1999.
45. Wesche H, Gao X, Li X, Kirschning CJ, Stark GR, Cao Z. IRAK-M is a novel member of the Pelle/interleukin-1 receptor-associated kinase (IRAK) family. J Biol Chem 274: 19403–19410, 1999.
46. Wesche H, Henzel WJ, Shillinglaw W, Li S, Cao Z. MyD88: an adapter that recruits IRAK to the IL-1 receptor complex. Immunity 7: 837–847, 1997.[CrossRef][Web of Science][Medline]
47. Yamin TT, Miller DK. The interleukin-1 receptor-associated kinase is degraded by proteasomes following its phosphorylation. J Biol Chem 272: 21540–21547, 1997.
48. Yao J, Kim TW, Qin J, Jiang Z, Qian Y, Xiao H, Lu Y, Qian W, Gulen MF, Sizemore N, DiDonato J, Sato S, Akira S, Su B, Li X. Interleukin-1 (IL-1)-induced TAK1-dependent Versus MEKK3-dependent NFkappaB activation pathways bifurcate at IL-1 receptor-associated kinase modification. J Biol Chem 282: 6075–6089, 2007.
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75 kDa) and modified IRAK-1 (100–250 kDa) bands. B: MEFs treated with LPS for 1 h were separated into plasma membrane (PM), cytosolic (Cyto), and nuclear (Nuc) enriched fractions by differential ultracentrifugation. Fractions were resolved by 10% SDS-PAGE and Western blots were probed for IRAK-1. Epidermal growth factor receptor (EGFR), GAPDH, and lamin B are markers for the plasma membrane, cytosolic, and nuclear enriched fractions, respectively.
