©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
NF-B Activation by Interleukin-1 (IL-1) Requires an IL-1 Receptor-associated Protein Kinase Activity (*)

Glenn E. Croston , Zhaodan Cao , David V. Goeddel (§)

From the (1)From Tularik, Inc., South San Francisco, California 94080

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

An important function of interleukin-1 (IL-1) is activation of the transcription factor NF-B, which is signaled via the type I IL-1 receptor (IL-1RI). By receptor mutagenesis studies, we have identified a region of the cytoplasmic domain of IL-1RI that is required for both IL-1-mediated NF-B activation and IL-1-dependent activation of a receptor-associated protein kinase activity we term IRAK. No IL-1RI mutants were found that can activate NF-B in the absence of IRAK activity. Therefore, we propose that IRAK activation is a necessary step in the activation of NF-B by IL-1.


INTRODUCTION

The cytokine interleukin-1 (IL-1)()is a key mediator in the inflammatory response (for reviews, see Refs. 1-3). The importance of IL-1 in inflammation has been demonstrated in animal models by the ability of the highly specific IL-1 receptor antagonist protein to relieve inflammatory conditions (for review, see Refs. 1 and 4). Many of the proinflammatory effects of IL-1, such as the up-regulation of cell adhesion molecules on vascular endothelia, are exerted at the level of transcriptional regulation. The transcriptional activation by IL-1 of cell adhesion molecules and other genes involved in the inflammatory response appears to be mediated largely by NF-B(5, 6, 7, 8) . In response to IL-1, the NF-B inhibitory factor IB is degraded, and NF-B is released from its inactive cytoplasmic state to localize within the nucleus where it binds DNA and activates transcription(9, 10) . Elucidation of the IL-1 signal transduction pathway leading to NF-B activation should provide valuable insight into potential mechanisms to alleviate inflammation.

Two cell surface IL-1 receptors, type I (IL-1RI) and type II (IL-1RII), have been identified and molecularly cloned(11, 12) . Both receptors have a single transmembrane domain and an IgG-like extracellular domain. The IL-1RII is found predominantly in B-cells, contains a cytoplasmic domain of only 29 amino acids, and may not play a direct role in intracellular signal transduction (for review, see Ref. 13). The human IL-1RI is found on most cell types and contains 552 amino acids in its mature form. Its cytoplasmic domain of 212 amino acids is required for signaling activity(14, 15, 16, 17) , but has no significant homology with protein kinases or any other mammalian factors involved in signal transduction. The cytoplasmic domain of IL-1RI does share significant sequence homology with the Drosophila transmembrane protein Toll that is involved in dorsal-ventral patterning(18) . This homology may be functionally significant since other components of the Drosophila dorsal-ventral patterning pathway, Dorsal and Cactus, are homologous with NF-B and IB, respectively(19) . Also, mutation of the amino acids that are conserved between IL-1RI and Toll inactivates IL-1RI signaling in T-cells(15) .

Here we describe the identification by deletion and site-directed mutagenesis of regions of the IL-1RI cytoplasmic domain required for IL-1-mediated NF-B activation. We have also identified by immunoprecipitation and in vitro kinase assays an IL-1-inducible protein kinase activity that associates with IL-1RI. Kinase activity is not detected in immunoprecipitates of IL-1RI mutants that are inactive in vivo. This IL-1 receptor-associated kinase (IRAK) may therefore play a central role in IL-1 signal transduction leading to NF-B activation.


EXPERIMENTAL PROCEDURES

Plasmid Construction and Antiserum Preparation

The human IL-1RI cDNA was cloned into pRK5 (20) to give the plasmid pRK-IL-1RI in which expression is under the control of the cytomegalovirus immediate early promoter-enhancer. Expression plasmids for the C-terminal deletion mutants of IL-1 receptor were generated from pRK-IL-1RI by introducing stop codons into the IL-1RI coding region by polymerase chain reaction (PCR). The internal triple mutants were made by a procedure involving two rounds of PCR. The first round of PCR generated overlapping fragments with the corresponding mutations in the center of the overlapped region. The two fragments were joined by a second round of PCR. The sequences of all constructs were confirmed by DNA sequencing. The pELAM-luciferase reporter plasmid containing E-selectin promoter sequences from -730 to +52 (21) was provided by Dr. Uli Schindler. The internal reference plasmid pRas-gal was provided by Dr. Jinzhao Hou. To prepare antiserum to the extracellular domain of the IL-1RI, a fusion protein consisting of the mature IL-1RI extracellular domain fused to human IgG as described (22) was expressed transiently in 293 cells. Cell culture medium containing the chimeric protein was harvested on days 3 and 7 after transfection. The IL-1RI-IgG fusion protein was purified by protein A-agarose chromatography and used to immunize rabbits by BAbCo (Richmond, CA).

Cell Culture, Transfection, Cell Extract Preparation, and Metabolic Labeling

Human embryonic kidney 293 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 100 µg/ml penicillin G, and 100 µg/ml streptomycin (Life Technologies, Inc.). To assay receptor function, cells were seeded in 6-well dishes at 30-50% confluence. Transfections were carried out the following day with the various expression plasmids by the calcium phosphate precipitation method(23) . 36 h later, human recombinant IL-1 (Genentech) was added to the medium at a final concentration of 1 ng/ml. The cells were harvested 6 h later and assayed for luciferase activity using Promega reagents. -Galactosidase activity was determined using chemiluminescent reagents (Tropix, Inc.) and used to normalize luciferase activities. Extracts for immunoprecipitations and in vitro phosphorylation assays were prepared as follows. 293 cells were seeded at 50% density in 100-mm plates and transfected with IL-1RI expression plasmids on the following day. 40 to 48 h later, IL-1 (20 ng/ml) was added to the medium. After incubation at 37 °C for the indicated times, medium was removed, and the plates were chilled on ice immediately. The cells were washed twice with 5 ml of ice-cold phosphate-buffered saline (PBS) and scraped off the plates in 5 ml of PBS containing 1 mM EDTA. Cells were pelleted by 1200 g centrifugation for 3 min and suspended in 1 ml of lysis buffer (50 mM HEPES, pH 7.6, 250 mM NaCl, 1 mM dithiothreitol, 1 mM EDTA, 0.1% Tween 20, 10% (v/v) glycerol, 10 mM -glycerophosphate, 5 mMp-nitrophenyl phosphate, 1 mM sodium orthovanadate, 1 mM benzamidine, 0.4 mM phenylmethylsulfonyl fluoride, 1 mM sodium metabisulfite, 10 µg/ml leupeptin, and 10 µg/ml aprotinin). After incubation on ice for 20 min, the cell debris was pelleted by a 20-min centrifugation in a microcentrifuge, and the supernatants were collected and stored at -70 °C. For metabolic labeling, 293 cells were seeded in 150-mm plates and grown to near-confluence. The cells were washed twice with 25 °C PBS and incubated with Dulbecco's modified Eagle's medium lacking cysteine and methionine at 37 °C for 40 min before addition of 700 µCi of S cell labeling mix (Amersham). 4 h later, the medium was removed, cells were washed twice with PBS, and extracts were prepared as described above.

Immunoprecipitation and in Vitro Kinase Assays

For immunoprecipitations, 1 ml of cellular extract was incubated with 20 µl of protein A-agarose slurry (50% v/v) in lysis buffer at 4 °C for 2 h. Protein A beads were pelleted by centrifugation in a microcentrifuge for 10 s, and 1 µl of rabbit antiserum or preimmune serum was incubated with the precleared supernatant at 4 °C for 2-3 h. The reactions were mixed with 20 µl of the protein A-agarose slurry and incubated for an additional 1 h. Protein A beads were collected by centrifugation in a microcentrifuge for 10 s and washed 5 times with 1 ml of lysis buffer. The beads were then suspended in 20 µl of kinase buffer containing 20 mM Tris-HCl, pH 7.6, 20 mM MgCl, 20 mM -glycerophosphate, 20 mMp-nitrophenyl phosphate, 1 mM sodium orthovanadate, 1 mM benzamidine, 0.4 mM phenylmethylsulfonyl fluoride, 1 mM sodium metabisulfite, 2 µM cold ATP, and 10 µCi of [-P]ATP. The kinase reactions were allowed to proceed at 30 °C for 30 min and terminated with 20 µl of SDS sample buffer. After boiling for 3-5 min, 20-µl reaction aliquots were separated by 8% SDS-polyacrylamide gel electrophoresis. Radiolabeled proteins were visualized by autoradiography.


RESULTS

Based on its lack of homology with any known mammalian signal transducers, it is likely that the intracellular region of IL-1RI interacts with other factors to transduce IL-1 signals. We sought to delineate a receptor domain that interacts with such factors by examining the ability of IL-1RI mutants to activate NF-B. To measure NF-B activation, we utilized an assay in which expression vectors for IL-1RI mutants were cotransfected with an E-selectin promoter-luciferase reporter plasmid into the human 293 cell line. Stimulation of E-selectin transcription by IL-1 is known to occur primarily through the activation of NF-B(24, 25) . Luciferase activity in transiently transfected 293 cells was determined in the presence or absence of IL-1 stimulation. In the absence of transfected receptor, IL-1 (1 ng/ml) induced a low level of transcriptional activation through endogenous IL-1RI (Fig. 1A). However, a large increase in IL-1 dependent transcriptional activation was observed in cells transiently transfected with wild type IL-1RI. This result demonstrates that the majority of reporter activity in transiently transfected cells is signaled by transfected IL-1RI and validates the use of this system for the analysis of IL-1RI mutants.


Figure 1: Activation of transcription factor NF-B by IL-1RI mutants. Human 293 cells were cotransfected with the pELAM-luc reporter plasmid and wild type (W.T.) or the various IL-1RI mutant expression vectors. The C-terminal truncation mutants of IL-1RI are shown in A, and the triple alanine mutants are shown in B. Luciferase activity was determined as described under ``Experimental Procedures'' in the presence and absence of 1 ng/ml IL-1.



Five different C-terminal truncation mutants of IL-1RI were examined for their ability to activate the E-selectin reporter in response to IL-1 (Fig. 1A). Removal of 20, 25, or 31 amino acids from the C terminus did not appreciably affect the ability of IL-1RI to activate NF-B. Deletion of 45 or 75 C-terminal amino acids eliminated the ability of IL-1RI to activate NF-B. Therefore, the region defined by the -31 and -45 deletions (residues 508-521) includes sequences required for the activation of NF-B by IL-1. Furthermore, the -45 and -75 deletion mutants behaved as dominant negative mutations and blocked the ability of the endogenous IL-1RI to activate NF-B.

Since amino acids 508 to 521 of IL-1RI appear necessary for signal transduction, this region was examined more closely by constructing receptors with sets of three amino acids mutated to alanine. These mutants, which include Ala-510-512, Ala-513-515, and Ala-518-520, were analyzed in the NF-B reporter assay for their ability to activate NF-B (Fig. 1B). By this analysis, the Ala-510-512 mutant is active, while the Ala-513-515 and Ala-518-520 mutants are inactive. Amino acids 510, 511, and 512 of the IL-1RI are not conserved in Toll, while conserved amino acids are present in both the 513-515 and 518-520 regions. The requirement of these conserved residues for IL-1RI function may indicate that these amino acids directly contact signaling molecules or are critical to overall receptor structure.

We next performed immunoprecipitation experiments to identify IL-1RI-associated signaling molecules. Immunoprecipitation of metabolically S-labeled IL-1RI from transiently transfected 293 cells reveals that the receptor is expressed at high levels and can be specifically immunoprecipitated with polyclonal antisera directed against the IL-1RI extracellular domain. The immunoprecipitating band at 80 kDa is mature IL-1RI and the 60-kDa band is the nonglycosylated receptor (Fig. 2A). In agreement with previously published results(20) , fluorescence-activated cell sorter analysis of 293 cells transiently transfected with IL-1RI indicated that a large percentage (40%) of the cell population expresses receptor (data not shown). The addition of IL-1 to cells prior to cell lysis had no effect on the ability of the antisera to immunoprecipitate IL-1RI (Fig. 2A, lanes 3 and 4).


Figure 2: Association of IL-1RI with an IL-1- inducible protein kinase activity. A, immunoprecipitation of transfected IL-1RI. 293 cells were transfected with the expression vector pRK-IL-1RI and metabolically labeled with [S]methionine and cysteine. Immunoprecipitations using either preimmune serum (lanes 1 and 2) or anti-IL-1RI antiserum (lanes 3 and 4) were performed as described under ``Experimental Procedures'' in the presence (lanes 2 and 4) or absence (lanes 1 and 3) of treatment with 20 ng/ml IL-1. B, in vitro kinase assay on immunoprecipitated IL-1RI. 293 cells were transfected with pRK-IL-1RI and incubated for 2 min in the presence (lanes 2 and 4) or absence (lanes 1 and 3) of IL-1. Immunoprecipitations using preimmune serum (lanes 1 and 2) or anti-IL-1RI serum (lanes 3 and 4) and in vitro kinase assays were performed as described under ``Experimental Procedures.'' C, time course of in vitro kinase activation by IL-1. 293 cells were transfected with pRK-IL-1RI and treated with IL-1 for the indicated times (minutes). Immunoprecipitations with anti-IL-1RI serum and in vitro kinase assays were performed as in B. Positions of size markers (kDa) are shown at the left.



To determine whether a protein kinase associates with IL-1RI, the receptor was immunoprecipitated from transiently transfected 293 cells and examined by an in vitro kinase assay. An IL-1-inducible protein kinase activity was observed that specifically associated with IL-1RI (Fig. 2B). We have termed this activity IRAK (IL-1RI-associated kinase). The major target of IRAK in this reaction is not the 80-kDa receptor, but an endogenous substrate of approximately 100 kDa. The specificity of the receptor-kinase interaction is supported by the absence of activity in the preimmune precipitate (Fig. 2B, lanes 1 and 2) and by the ability of an IL-1RI-IgG fusion protein to compete away the kinase activity when added to the immunoprecipitation (data not shown). Kinase activation occurred rapidly, reaching an optimum within 2 min of exposure of cells to IL-1, suggesting that activation of the kinase occurs proximally to the IL-1 receptor (Fig. 2C).

If IRAK is involved in NF-B activation, then the activity of the kinase in immunoprecipitates of mutated receptor should correlate with in vivo activation of the E-selectin reporter by mutated receptors. The C-terminal deletion mutants of IL-1RI were transiently expressed in 293 cells and receptor was immunoprecipitated and examined for associated IL-1-inducible kinase activity (Fig. 3A). In the absence of transfected receptor, 293 cells display low but detectable levels of IRAK activity (lane 2). All three C-terminal deletion mutants(-20, -25, -31) that can activate NF-B display associated kinase activity that is indistinguishable from that associated with intact IL-1RI. IRAK activity does not coprecipitate with the -45 deletion mutant that was unable to activate NF-B. Thus, there is a direct correlation between the association of active IRAK with IL-1RI and the ability of IL-1 to activate NF-B.


Figure 3: Activation of IRAK by IL-1RI mutants. 293 cells were transfected with pRK-IL-1RI (wild type) or expression plasmids for the various IL-1RI mutants. Immunoprecipitations and in vitro kinase assays were performed as described in the legend to Fig. 2B. Positions of size markers (kDa) are shown at the left.



To further examine the connection between NF-B activation and IRAK kinase activity, the triple alanine scan mutants of IL-1RI were examined by the coimmunoprecipitation assay following transfection into 293 cells (Fig. 3B). IRAK activity was observed with the Ala-510-512 mutant, but not with the Ala-513-515 or Ala-518-520 mutants (lanes 9-12). Once again there was a direct correlation between the ability of an IL-1RI mutant to interact with IRAK and to induce NF-B activation.


DISCUSSION

Many of the biological properties of IL-1 are a consequence of the activation of the transcription factor NF-B, which subsequently induces expression of many genes. IL-1-mediated activation of NF-B is signaled by IL-1RI, the type I cell surface receptor for IL-1(17, 26) . However, very little is known about the post-receptor signaling mechanisms of IL-1RI.

Previous studies have shown that the intracellular domain of IL-1RI is necessary for IL-1-mediated prostaglandin and granulocyte-colony stimulating factor synthesis(14) , IL-8 gene induction(16) , and IL-2 promoter activation(15) . The cytoplasmic domain of IL-1RI contains sequence similarity to the Drosophila Toll protein involved in dorsal-ventral patterning. Since the transduction of the Toll signal to the Drosophila nucleus requires activation of the protein kinase Pelle, it is possible that IL-1RI signaling pathways will also utilize one or more protein kinases. In this regard, Martin et al.(27) , using exogenous substrates, recently demonstrated the existence of an IL-1-dependent serine/threonine protein kinase activity that co-precipitates with IL-1RI. We have also identified an IL-1RI-associated kinase, IRAK, whose phosphorylation of an endogenous substrate of 100 kDa is dependent on IL-1. However, the relationship of these two activities to each other cannot be ascertained at this time.

We have confirmed and extended these earlier studies to show a direct correlation between NF-B activation and an IL-1-dependent, IL-1RI-associated protein kinase activity. By mutational analysis, we showed that the C-terminal 31 amino acids of IL-1RI are dispensable for the signaling of both activities. However, deletion of an additional 14 amino acids inactivates both responses. We then performed alanine scan mutagenesis on three groups of three amino acids in this region. Two of these mutant IL-1RIs (Ala-513-515 and Ala-518-520) were unable to coprecipitate IRAK activity or signal NF-B activation. The mutations in both of these mutant receptors are in residues that are conserved between Toll and IL-1RI. Previously, Heguy et al.(15) had shown that these conserved residues are required for induction of IL-8 gene expression.

The identification of a protein kinase activity that associates with IL-1RI, and whose activity is dependent on IL-1, provides an initial step in the signal transduction pathway(s) mediated by IL-1. The strict concordance between IRAK and NF-B activity for the various IL-1RI mutants suggests that IRAK activity may be required for IL-1-mediated NF-B activation. It will now be of great interest to determine whether IRAK directly phosphorylates IB, leading to its degradation and release from NF-B, or activates a protein kinase cascade that subsequently targets IB.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed: Tularik, Inc., 270 E. Grand Ave., South San Francisco, CA 94080. Tel.: 415-615-4200; Fax: 415-615-4222.

The abbreviations used are: IL-1, interleukin-1; IRAK, IL-1 receptor-associated kinase; PCR, polymerase chain reaction; PBS, phosphate-buffered saline.


ACKNOWLEDGEMENTS

We thank Keith Williamson for DNA sequencing and Dean Stott for synthetic DNA.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.