From the
An important function of interleukin-1 (IL-1) is activation of
the transcription factor NF-
The cytokine interleukin-1 (IL-1)
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-
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-
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-
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-
We next performed immunoprecipitation
experiments to identify IL-1RI-associated signaling molecules.
Immunoprecipitation of metabolically
If
IRAK is involved in NF-
Many of the biological properties of IL-1 are a consequence
of the activation of the transcription factor NF-
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
We have confirmed and extended these
earlier studies to show a direct correlation between NF-
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-
We thank Keith Williamson for DNA sequencing and Dean
Stott for synthetic DNA.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
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.
(
)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 I
B 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.
B
and I
B, respectively(19) . Also, mutation of the amino
acids that are conserved between IL-1RI and Toll inactivates IL-1RI
signaling in T-cells(15) .
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.
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.
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.
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.
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).
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.
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.
100 kDa is dependent on IL-1. However,
the relationship of these two activities to each other cannot be
ascertained at this time.
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.
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 I
B, leading to its degradation and release
from NF-
B, or activates a protein kinase cascade that subsequently
targets I
B.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.