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INTRODUCTION |
The innate immune response is rapidly activated upon exposure to
environmental stimuli and discriminates between self and nonself. The
innate immune response may also be involved in determining whether an
acquired immune response is required following pathogenic invasion (1).
Studies in plants, insects, and mammals have revealed that
serine/threonine innate immune kinase family members are components of
the innate immune response (2). Serine/threonine innate immune kinase
mediate early developmental decisions and, in adult tissue, mediate
transactivation of genes whose products are involved in host defense. A
Drosophila serine/threonine innate immune kinase family
member, Pelle, is a maternal-effect gene that is also required for
protection against fungal infections (2, 3). Plant serine/threonine
innate immune kinase family members include Pto and Pti-1, which
mediate disease resistance in the tomato (4). Two human
serine/threonine innate immune kinase family members,
interleukin-1 receptor associated
kinases IRAK and IRAK-2 have been linked to signaling
through IL-11 receptor family
members (5-7).
In response to IL-1 binding, the type I IL-1 receptor recruits the
IL-1 receptor accessory
protein (IL-1RAcP) and MyD88 (for review, see Ref. 8). The
IL-1RAcP and/or MyD88 bind IRAK (9-11) and in a tumor
necrosis factor receptor-associated factor-6 (TRAF-6)-dependent manner activate the
NF-
B-inducing kinase (NIK; Refs.
12 and 13). NIK promotes activation of the I
B kinase complex
(14-18), culminating in activation of NF-
B.
IRAK was identified by co-purification with the IL-1RI and was
subsequently shown to bind the IL-1RAcP directly (5, 6, 9-11). IRAK-2,
like IRAK, is in a complex with IL-1RI and IL-1RAcP and is a component
of a transduction pathway downstream of MyD88 (6). IRAK or IRAK-2
overexpression activates the NF-
B-dependent E-selectin
gene promoter (6, 11). In response to IL-1 stimulation, the IRAK
protein becomes phosphorylated (5, 19). Whether IRAK phosphorylation is
an autophosphorylation event or the result of phosphorylation by an
independent kinase is not known. Nor is it known whether IRAK
phosphorylation is an activation event and/or is required for
IL-1-targeted degradation of IRAK by the proteosome (19). Two types of
IRAK and IRAK-2 mutants have been described: mutants that consist of
the amino terminus of IRAK or IRAK-2, and for IRAK, a mutant in which a
lysine in the putative ATP binding site has been changed to serine
(IRAKK239S). Overexpression of the amino terminus of IRAK or IRAK-2
inhibits IL-1-dependent activation of NF-
B activity,
thus implicating IRAK and IRAK-2 in IL-1 signaling (6, 7, 11, 20). The
effect of IRAKK239S overexpression on IL-1-dependent
NF-
B activation has not been reported.
We identified a mouse homologue of Pelle, a Drosophila
serine/threonine innate immune kinase family member (21). Based upon sequence identity (5) and chromosomal location (22), mPLK is the mouse
homologue of human IRAK. The mPLK protein contains intrinsic protein
kinase activity (21). Although IRAK and IRAK-2 share sequence
similarity, IRAK-2 lacks key residues thought to be critical for
protein kinase activity.2
This led us to question whether mPLK/IRAK protein kinase catalytic activity is essential in NF-
B activation and IL-1 signaling. Dominant-negative alleles of serine/threonine protein kinases have been
generated either by mutating the lysine in protein kinase subdomain II
or the aspartic acid in protein kinase subdomain VII (23). In
mPLK/IRAK, these sites correspond to amino acid residues Lys-239 and
Asp-358, respectively. Because mutation of the corresponding lysine
residue in other kinases does not inevitably result in loss of
catalytic activity (24), we generated a catalytically inactive kinase
by mutating the aspartic acid in protein kinase subdomain VII of
mPLK/IRAK. We show here that introduction of a D358N mutation in
subdomain VII of mPLK/IRAK abrogates the ability of mPLK/IRAK to induce
NF-
B activity. Furthermore, mPLK/IRAKD358N functions as a
dominant-negative allele because it inhibits wild-type mPLK/IRAK
activity. We have used this mutation to confirm a role for mPLK/IRAK in
IL-1 signaling and to identify a role for mPLK/IRAK in the TNF RI
signaling pathway.
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EXPERIMENTAL PROCEDURES |
Plasmid Construction and Plasmids--
The IL-8-CAT and IL-8-LUC
reporter constructs contained bp
1451-+44 of the IL-8 gene (25)
immediately upstream of either the bacterial chloramphenicol
acetyltransferase gene (pCAT-Basic, Promega, Madison, WI) or the
firefly luciferase cDNA (pGL3, Promega). The indicated mPLK
cDNAs were subcloned into a mammalian expression vector that placed
them under the control of the cytomegalovirus immediate-early gene
promoter (pCMV). The mPLK construct contained the wild-type mPLK
cDNA (amino acids 1-711; Ref. 21). The
mPLK cDNA encoded
amino acids 33-711. The cimPLK mutant contained a point mutation at
amino acid 358 (D358N) and, in in vitro kinase assays,
lacked catalytic activity.2 The NIK plasmid construct
contained the wild-type NIK cDNA, and ciNIK encoded NIKKK429-430
amino acids (13). TRAF2 encoded the wild-type TRAF2 cDNA,
TRAF2
encoded amino acids 87-501 (26), TRADD encoded the wild-type TRADD
cDNA, and
TRADD encoded amino acids 102-312 (27). The
full-length mPLK cDNA was tagged with a myc epitope at the carboxyl
terminus (Invitrogen, San Diego, CA); full-length NIK cDNA
contained a carboxyl-terminal FLAG epitope. Epitope-tagged mPLK and NIK
constructs were determined to be biologically active in preliminary
studies).2
Cell Culture and Transfections--
Human embryonic kidney
epithelial cells (293 cell line) were maintained in Dulbecco's
modified Eagle's medium containing 10% fetal bovine serum,
penicillin/streptomycin, and glutamine. The C3H10T1/2 mouse embryo
fibroblast cell line was maintained in Basal's modification of
Eagle's media supplemented with glutamine, penicillin/streptomycin,
and 10% fetal bovine serum. For transient transfection assays,
C3H10T1/2 mouse embryo fibroblasts were plated at a density of 2 × 105 cells/60-mm dish, and
Ca3(PO4)2 precipitates were used to
introduce plasmid constructs into cells as described previously (28). To monitor transfection efficiency, precipitates also included a
reporter construct containing the luciferase gene under control of the
herpes simplex virus thymidine kinase promoter (Tk-LUC; ATCC, Manassas,
VA) or the
-galactosidase gene under control of the Rous sarcoma
virus LTR (RSV-
GAL; Ref. 29). Cultures were harvested 24 or 48 h after transfection. Individual assays were normalized for luciferase
activity when IL-8-CAT reporter construct activity was examined or were
normalized for
-galactosidase activity when IL-8-LUC reporter
construct activity was examined. Luciferase (Promega, Madison, WI) and
-galactosidase (TROPIX, Inc., Bedford, MA) activities were assayed
according to the manufacturer's specifications, and data are presented
as a ratio between the IL-8-CAT and Tk-LUC or IL-8-LUC and RSV-
GAL.
Data are from two to three independent experiments performed in
duplicate or triplicate with similar qualitative results.
Antibodies, Immunoprecipitation, and Western
Blotting--
Monoclonal mouse anti-c-myc antibody was purchased from
Roche Molecular Biochemicals. The anti-FLAG M2 monoclonal antibody was
purchased from Eastman Kodak Co. Chromatographically purified mouse IgG
was purchased from Zymed Laboratories Inc.
Laboratories (South San Francisco). Bacterially expressed HIS/T7-tagged
mPLK was used as an immunogen to raise polyclonal antisera in rabbits (HRP Inc., Denver, PA). Crude antisera were affinity-purified using
denatured mPLK protein immobilized on Immobilon-P membrane (Millipore).
The C3H10T1/2 mouse embryo fibroblast cell line was used in the
mPLK-TNF RI co-immunoprecipitation assays. Approximately 5 × 105 cells were seeded into 100-mm tissue culture dishes
24 h before treatment with recombinant mouse TNF
(100 units/ml)
for 5 or 15 min. Cell monolayers were harvested, and immunocomplexing
assays were performed as described below. For the mPLK-NIK
co-immunoprecipitation assays, the human embryonic kidney epithelial
cells (293 cell line; ATCC) were used. Approximately 2 × 106 cells were seeded into 60-mm tissue culture dishes and
grown in 5% CO2 at 37 °C. Plasmid constructs encoding
myc-tagged mPLK and FLAG-tagged NIK were transfected into cells 16 h later by calcium phosphate precipitation (28). 48 h later, cell
monolayers were harvested.
Cell monolayers were rinsed with phosphate-buffered saline and lysed in
1 ml of immunoprecipitation (IP) lysis buffer (10 mM HEPES,
pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% Triton
X-100, and complete protease inhibitors). Cell debris was removed by
centrifugation, and cell lysates were incubated with the indicated
immunoprecipitation antibody. After a 16-h incubation at 4 °C,
protein A-Sepharose (10% (v/v) slurry) was added to
antibody-containing cell lysates, and reactions were subject to an
additional 2-h incubation at 4 °C. Immunocomplexes, collected by
centrifugation, were washed two times in IP wash buffer (same as lysis
buffer, except [Triton X-100] was 0.1%). Washed material was
resuspended in Laemmli buffer, denatured, and subjected to
SDS-polyacrylamide gel electrophoresis in 8% reducing polyacrylamide
gels. Separated proteins were transferred to Immobilon-P (Millipore)
for Western analysis.
Membranes were blocked and probed with the indicated primary antisera
and HRP-conjugated secondary antibody in KPBS-T (140 mM
NaCl, 2.7 mM KCl, 8 mM
Na2HPO4, 1.5 mM
KH2PO4, 0.4% Tween 20) containing 5% (w/v)
nonfat dry milk. Interactions were visualized with ECL (Amersham
Pharmacia Biotech) according to manufacturer's specifications.
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RESULTS |
mPLK/IRAK Activates NF-
B--
Treatment of a variety of cell
types with inflammatory cytokines, like IL-1
and TNF
, results in
transactivation of genes involved in mediating immune and inflammatory
responses, including the IL-8 and E-selectin genes (25, 30).
Overexpression of human IRAK induces transactivation of the
NF-
B-dependent E-selectin gene promoter (11). Therefore
we first confirmed that overexpression of mPLK, the mouse IRAK
homologue, transactivates an NF-
B-dependent gene
promoter in mouse cells. Transient transfection of mouse embryo
fibroblasts with a mammalian expression vector containing the mPLK
cDNA led to approximately a 5-fold increase in the activity of the
NF-
B-dependent IL-8 gene promoter (Fig.
1A). This level of
mPLK-mediated induction of NF-
B activity is comparable with that
detected when IRAK is overexpressed in human cells (11). These data are
consistent with the proposal that mPLK, like its human homologue, IRAK,
lies in a signaling pathway upstream of NF-
B. Substitution of an
asparagine for an aspartic acid residue (D358N) in the
Mg2+-ATP binding site of mPLK created catalytically
inactive mPLK protein (cimPLK).2 In contrast to results
obtained with mPLK, overexpression of cimPLK did not result in
transactivation of the IL-8 gene promoter (Fig. 1A). A
catalytically active mPLK mutant lacking the first 33 amino acids
corresponding to helix 1 of the putative mPLK/IRAK death domain
(
mPLK; Ref. 21) also did not transactivate the IL-8 gene promoter
(Fig. 1A). Thus, mPLK/IRAK catalytic activity and an intact
amino-terminal death domain are required for mPLK to induce
transactivation of the IL-8 gene promoter. To determine whether cimPLK
functions in a dominant-negative manner, mouse embryo fibroblasts were
co-transfected with mPLK and cimPLK. In a dose-dependent
manner, cimPLK inhibited the ability of mPLK to induce IL-8 promoter
activity (Fig. 1B). Similar results were obtained with
mPLK.2

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Fig. 1.
mPLK activation of
NF- B-dependent promoters.
A, mPLK transactivates the IL-8 gene promoter. Mouse embryo
fibroblasts were cotransfected with an IL-8-LUC construct (2.5 µg)
and 5.0 µg of the indicated constructs. See "Experimental
Procedures" for transfection and enzyme assay methods. B,
catalytically inactive mPLK functions as a dominant negative. Mouse
embryo fibroblasts were cotransfected with an IL-8-CAT reporter
construct (5 µg) and the indicated plasmid constructs. C,
mPLK transactivates the E-selectin gene promoter. Mouse embryo
fibroblasts were cotransfected with an E selectin-LUC reporter
construct (2.5 µg) and the mPLK cDNA construct (5 µg);
D, mPLK does not transactivate the
AP-1-dependent IL-11 gene promoter. Mouse embryo
fibroblasts were cotransfected with an IL-11-LUC reporter construct
(2.5 µg) and 5.0 µg of the mPLK construct. The indicated culture
was treated with TNF for 16 h before harvest. Transfectants
were harvested and processed as described under "Experimental
Procedures."
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The IL-8 gene promoter, like the E-selectin promoter, contains NF-
B
and AP1 cis-acting elements (25, 31). Thus, the observations described
above do not define which of these cis-acting elements is activated by
mPLK. To verify that the mPLK effect is mediated through NF-
B, the
effect of mPLK overexpression on NF-
B-dependent promoters was examined. Like the IL-8 gene promoter, overexpression of
mPLK increased E-selectin promoter activity (4-fold; Fig.
1C) and stimulated the activity of a reporter construct
under the control of tandem NF-
B sites (see Fig. 3C; Ref.
32). Because the E-selectin and IL-8 promoters also contain binding
sites for AP1 family members, we examined whether mPLK affected the
activity of an IL-11 promoter construct known to be
AP1-dependent (33). Although TNF
treatment led to a
4-fold increase in IL-11 promoter activity, overexpression of mPLK had
no effect on IL-11 promoter activity (Fig. 1D). These data
demonstrate that mPLK/IRAK lies in a signaling pathway upstream of
NF-
B.
The NF-
B-inducing kinase, NIK, is a component of the type I IL-1
receptor (IL-1RI)-signaling cascade leading to NF-
B activation (13).
We therefore determined whether induction of the IL-8 gene promoter by
mPLK required NIK. Transfection of mouse embryo fibroblasts with NIK
led to an approximate 18-fold increase in IL-8 gene promoter activity
(Fig. 2). Co-transfection with cimPLK or
mPLK had no effect upon NIK-mediated activation of the IL-8 gene
promoter (Fig. 2). Malinin et al. (13) describe a
dominant-negative allele of NIK, ciNIK (K429A,K430A), capable of
blocking IL-1 or TNF
-induced transactivation of the E-selectin gene
promoter. However this construct has no effect on p65-induced
E-selectin gene promoter activity. We examined whether ciNIK
overexpression would affect the ability of mPLK to induce IL-8 gene
promoter activity. Results of these assays suggested that
mPLK/IRAK-mediated transactivation of the IL-8 gene promoter requires
NIK (Fig. 2).

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Fig. 2.
mPLK is upstream of NIK. Mouse embryo
fibroblasts were cotransfected with the IL-8-LUC reporter construct and
the indicated plasmid constructs. Transfectants were harvested and
processed as described under "Experimental Procedures."
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IL-1 Signaling Does Not Require mPLK/IRAK Activity--
NIK and
mPLK/IRAK are thought to mediate activation of NF-
B through the
IL-1RI (11, 13). IRAK-2, which lacks conserved residues in key protein
kinase subdomains, can also modulate IL-1-induced NF-
B activation
(6). Therefore we determined whether mPLK/IRAK catalytic activity is
necessary for IL-1-dependent activation of the IL-8 gene
promoter. Overexpression of wild-type mPLK did not block IL-1 induction
of IL-8 gene promoter activity (Fig. 3A). Interestingly, neither
cimPLK nor
mPLK decreased IL-1 induction of IL-8 promoter activity
(Fig. 3A). We therefore examined whether the link between
IL-1 and mPLK/IRAK activity in mouse embryo fibroblasts was similar to
that described for mPLK/IRAK activity in human cells. Overexpression of
amino-terminal IRAK (amino acids 1-208 or 1-215) or IRAK-2 (amino
acids 1-96) mutants inhibit IL-1-dependent NF-
B
activation in human cells (6, 7, 11). Therefore we prepared an mPLK
mutant that contained only amino-terminal residues 1-156.
Overexpression of the mPLK mutant containing only amino-terminal
residues 1-156 in mouse embryo fibroblasts blocked IL-1-dependent NF-
B activation.2 This result
is consistent with that described for amino-terminal IRAK mutants
expressed in human cells (6, 7, 11) and suggests that mPLK activity in
mouse cells functions in a manner similar to that proposed for IRAK in
human cells. However, when mouse embryo fibroblasts transfected with
dominant-negative mPLK/IRAK were treated with IL-1
for varying
times, higher levels of IL-8 promoter activity were detected as
compared with IL-1-treated fibroblasts transfected with vector alone
(Fig. 3B). To confirm that the enhanced response in
IL-1-treated cultures overexpressing cimPLK was mediated through
NF-
B, transient transfection assays were repeated with a reporter
construct containing only NF-
B sites ([NF-
B]3-LUC;
Ref. 32). Trace amounts of [NF-
B]-LUC activity were detected in
cultures transfected with either expression vector minus a cDNA
insert or with cimPLK. As was seen for IL-8 promoter activity, a 6-h
IL-1 treatment increased [NF-
B]3-LUC activity 2-fold,
and in cultures transfected with cimPLK, IL-1 treatment increased
[NF-
B]3-LUC activity 4-fold. Thus, it appears that
NF-
B-dependent signaling through the IL-1RI can be
blocked by overexpressing amino-terminal mPLK/IRAK residues or enhanced by overexpressing catalytically inactive mPLK/IRAK.

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Fig. 3.
mPLK activity required for TNF RI-induced
NF- B activity. A,
dominant-negative mPLK proteins do not block IL-1 -mediated
transactivation of the IL-8 gene promoter. Mouse embryo fibroblasts
were co-transfected as described above (Fig. 2B). 16 h
before harvest, cultures were treated with recombinant human IL-1
(3000 units; a kind gift from Hoffman-LaRoche Inc., Nutley, N.J.).
B, dominant-negative mPLK enhances IL-1 -mediated
transactivation of the IL-8 gene promoter. Mouse embryo fibroblasts
were transfected with cimPLK (5 µg) and IL-8-LUC (5 µg), and at the
indicated times before harvest, cultures were treated with recombinant
human IL-1 . C, dominant-negative mPLK blocks
TNF -dependent transactivation of the IL-8 gene promoter.
Mouse embryo fibroblasts were cotransfected with 2.5 µg of the
IL-8-LUC reporter construct and 5 µg of the indicated plasmid
constructs. 16 h before harvest, cultures were treated with
recombinant mouse TNF (100 units; Sigma). The effect of the ciNIK
construct on TNF -induced activation of the IL-8 gene promoter was
included as a positive control for monitoring inhibitory
activity.
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TNF RI Signaling Is mPLK/IRAK-dependent--
NIK also
mediates activation of NF-
B through the TNF RI, which contains a
death domain that associates with other death domain-containing proteins during signaling (13, 34, 35). The presence of a death domain
motif in mPLK/IRAK (21) suggested that mPLK/IRAK may be a component of
the TNF
as well as the IL-1-signaling pathways. Treatment of mouse
embryo fibroblasts with TNF
results in approximately a 30-fold
increase in IL-8 gene promoter activity (Fig. 3C). When mouse embryo fibroblasts transfected with mPLK are treated with TNF
,
no additional increase in IL-8 gene promoter activity is observed (Fig.
3C). However, when mouse embryo fibroblasts are transfected
with cimPLK or
mPLK, TNF
-induced transactivation of the IL-8 gene
promoter is decreased (Fig. 3C). The cimPLK- and
mPLK-mediated decrease in TNF
-induced activation of the IL-8 gene
promoter was dose-dependent; however, overexpression of
either cimPLK or
mPLK did not completely abrogate a
TNF-dependent signal.2 Analysis of cells
derived from TNF RI and TNF RII nulligenic animals revealed that
TNF-induced NF-
B activity is mediated solely through TNF RI (36).
Consistent with this observation, cimPLK decreased IL-8 promoter
activity in mouse fibroblasts treated with human TNF
,2
which on mouse cells signals exclusively through TNF RI (37). Taken
together, these data demonstrate that mPLK/IRAK is a component of a TNF
RI signaling pathway and provide further evidence for mPLK/IRAK as a
component of the IL-1-signaling pathway.
IL-1-mediated activation of NF-
B-dependent promoters is
inhibited by a mutated version of TRAF6 (11, 12). We found that dominant-inhibitory TRAF6 (
TRAF6289-522) inhibits
IL-1-dependent activation of the IL-8 gene promoter 10-fold
but did not significantly inhibit TNF- or mPLK-dependent
activation (1.4-fold decrease under either condition).2
These results support those of Cao and co-workers (11, 12), whereby
similar amounts of the dominant-inhibitory TRAF6 reduced an
IL-1-dependent signal 10-fold and reduced TNF- and the
IRAK-dependent signals 1.5-fold.
TNF-induced NF-
B activity is mediated in part by TNF
RI-associated death
domain (TRADD) and two TRADD-recruited proteins: TNF receptor associated
factor-2 (TRAF2) and the TNF
receptor-interacting protein (RIP;
Refs. 38-41). To determine whether mPLK/IRAK is required for
TRADD/TRAF2/RIP-mediated activation of the IL-8 gene promoter, the
effects of cimPLK and
mPLK on TRADD and TRAF2-induced IL-8 gene
promoter activity were measured. Neither cimPLK nor
mPLK blocked the
ability of TRADD (Fig. 4B) or
TRAF2 (Fig. 4A) to induce activation of the IL-8 gene
promoter.

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Fig. 4.
mPLK activity is
TRADD/TRAF2-independent. A, cimPLK or mPLK do not
block TRAF2-mediated transactivation of the IL-8 gene promoter. Mouse
embryo fibroblasts were cotransfected with 2.5 µg of the IL-8-LUC and
the indicated plasmid constructs. B, neither cimPLK nor
mPLK blocks TRADD transactivation of the IL-8 gene promoter. Mouse
embryo fibroblasts were cotransfected with 2.5 µg of the IL-8-LUC, 4 µg of a mammalian expression vector encoding the cowpox CrmA gene to
prevent TRADD-induced apoptosis (6). C, neither TRAF2 nor
TRADD interfere with mPLK activity. Mouse embryo fibroblasts were
cotransfected with (NF- B)3-LUC (2.5 µg) and the
indicated plasmid constructs.
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We next determined if TRADD or TRAF2 were required for
mPLK/IRAK-mediated activation of NF-
B. In contrast to mPLK, which does not transactivate AP1-dependent promoters (Fig.
1D), TRAF2 activates AP1 as well as
NF-
B-dependent promoters (42). The IL-8 gene promoter,
like the E-selectin gene promoter, contains AP1 and NF-
B sites.
Therefore, the requirement for TRADD and/or TRAF2 in mPLK/IRAK
signaling was evaluated with a reporter construct that only contains
NF-
B sites ([NF-
B]3-LUC; Ref. 32). Neither
TRADD
nor
TRAF2, which decrease NF-
B activation through TNF RI and TNF
receptor family members (34, 43), blocked mPLK-mediated activation of
the NF-
B-dependent reporter (Fig. 4C).
Transfection of mPLK with wild type or mutant TRAF2 (or TRADD) had an
additive effect on induction of NF-
B-dependent (Fig.
4C) as well as IL-8 gene promoter activity (Fig. 4,
A and B). A similar additive effect was detected
when wild type or dominant-negative mPLK constructs were co-transfected
with wild-type TRADD (or TRAF2; Fig. 4, A-C). These data
indicate that mPLK/IRAK lies in a TRADD/TRAF2-independent signaling pathway.
TNF RI signaling in part is thought to be mediated by the recruitment
of death domain-containing proteins, which bind to the carboxyl-terminal death domain in TNF RI (13, 34, 35). Because mPLK
contains a death domain and overexpression of cimPLK decreases IL-8
promoter activity in mouse fibroblasts treated with human TNF
,2 which signals exclusively through TNF RI (37), we
next determined whether the mPLK protein associates with TNF RI. TNF RI
anti-sera was used to immunoprecipitate proteins from cell lysates
prepared from mouse embryo fibroblasts that had been treated with
TNF
for 5 or 15 min. SDS-polyacrylamide gel electrophoresis followed by Western blot analysis of the immunocomplexes revealed the presence of mPLK protein in the immunocomplexes generated with the TNF RI
antisera in unstimulated cells. Furthermore the amount of mPLK protein
associated with TNF RI appeared to increase in response to TNF
treatment (Fig. 5). When mouse embryo
fibroblasts were transfected with myc-tagged cimPLK, immunocomplexes
generated with TNF RI anti-sera contained cimPLK (data not presented).
Finally, similar studies were performed in the human 293 cell line, and myc-tagged mPLK was found in TNF RI-containing complexes (data not
presented). Because cimPLK did not affect NIK activity (Fig. 2), these
results suggest the ability of cimPLK to block a TNF signal is most
likely mediated at the level of the TNF RI receptor.

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Fig. 5.
Endogenous mPLK and TNF RI
co-precipitate. Cell lysates were prepared from control,
nontreated mouse embryo fibroblasts or mouse embryo fibroblasts treated
with recombinant mouse TNF (100 units/ml) for 5 or 15 min. Cellular
proteins immunocomplexed with control mouse IgG or with TNF RI antisera
were separated by SDS-polyacrylamide gel electrophoresis, Western blots
were prepared and probed with mPLK or TNF RI antisera (see
"Experimental Procedures" for details).
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mPLK/IRAK and TRAF family members function upstream of NIK (13, 34).
The WKI motif found in TRAF proteins is required for binding to NIK
(34). Because mPLK/IRAK contains a similar motif (WHL; Refs. 5 and 21),
we postulated that the mPLK/IRAK and NIK proteins may complex. To test
this hypothesis, human embryonic kidney epithelial cells (293 cell
line) were co-transfected with plasmids encoding myc-tagged mPLK and
FLAG-tagged NIK. Myc or FLAG antisera were used to immunoprecipitate
proteins from cell lysates. Immunoprecipitated proteins were subjected
to SDS-polyacrylamide gel electrophoresis, followed by Western blot
analysis. mPLK and NIK were detected in immunoprecipitates generated
with either the FLAG or the myc antisera (Fig.
6, A and B).
However, neither mPLK nor NIK were in immunocomplexes prepared with an
unrelated mouse IgG (Fig. 6, A and B, lanes
1 and 2, respectively). These data indicate that
mPLK/IRAK and NIK can complex in cells.

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Fig. 6.
mPLK complexes with NIK.
Co-immunoprecipitation of NIK with mPLK.
Ca3(PO4)2 precipitates were used to
transfect 293 cells with plasmid constructs encoding myc-tagged mPLK (5 µg) and FLAG-tagged NIK (5 µg). 48 h later, cell lysates were
prepared, and immunoprecipitates were isolated with the indicated
amounts of purified mouse IgG, anti-myc, or anti-flag antisera. Western
blots containing the immunoprecipitated proteins were prepared and
probed with an anti-myc monoclonal antibody (A) or an
anti-FLAG monoclonal antibody (B) (see "Experimental
Procedures" for details).
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DISCUSSION |
mPLK/IRAK has been linked to signaling through IL-1 receptor
family members (5-7) and has been shown to have protein kinase activity in vitro (21). However, the importance of mPLK/IRAK protein kinase activity in signaling has not been addressed.
Frequently, mutations within the ATP binding site of protein kinases
not only encode nonfunctional protein kinases but also interfere with
the function of the wild-type protein (23). Thus, this mutation can be
described as a dominant negative allele of mPLK/IRAK (44) and will be
useful for further dissection of mPLK/IRAK function. We report here
that an mPLK/IRAK mutant lacking catalytic activity (D358N) was unable
to induce the activity of NF-
B-dependent promoters. Furthermore, the D358N mPLK/IRAK mutant decreased the ability of
wild-type mPLK/IRAK to activate an NF-
B-dependent
promoter in a dose-dependent fashion.
Interestingly, overexpression of cimPLK does not inhibit the ability of
IL-1 to induce the activity of an NF-
B-dependent promoter. In fact, overexpression of cimPLK enhances an
IL-1-dependent signal. This result suggests that mPLK/IRAK
catalytic activity is not required for its role in the IL-1-signaling
pathway. IRAK-2 has been described as a relative of IRAK that can also
enhance an IL-1 signal (6). IRAK-2 is quite similar to IRAK; however, it lacks key residues in several of the highly conserved protein kinase
subdomains and is unlikely to be catalytically active. Thus cimPLK may
enhance an IL-1 signal by mimicking IRAK-2. In this context,
independent of catalytic activity, IRAK2 and/or cimPLK may subserve a
scaffolding function and facilitate formation of signaling complexes.
Alternatively, in response to IL-1, phosphatidylinositol 3-kinase
activity is also increased (45), which, independent of IRAK/mPLK,
may effect changes in NF-
B-dependent signaling.
Enhancement of the IL-1-dependent signal in the presence of
cimPLK/IRAK suggests mPLK/IRAK catalytic activity may negatively regulate IL-1-initiated signaling. mPLK/IRAK mutants that inhibit or
enhance an IL-1-dependent signal may have therapeutic
utility. Clearly, identification of targets that can be used to block
IL-1-dependent signaling is important for down-regulating
inflammatory responses. As important, however, may be the
identification of targets that enhance/activate an inflammatory
response in an otherwise immunocompromised host.
TRAF6 has been linked to IL-1 and mPLK/IRAK signaling (11, 12).
Although overexpression of a dominant-inhibitory TRAF6 mutant decreases
IL-1 signaling, this mutant has a weaker inhibitory effect on
IRAK/mPLK. In fact, mutant TRAF6 interferes with IRAK/mPLK and TNF
similarly. Our own observations have confirmed these findings. Thus the
inhibitory effect of the TRAF6 mutant is much more robust in the
context of the IL-1-signaling pathway than in the mPLK/IRAK or TNF RI
signaling pathways. Interestingly, TRAF6 was recently shown to complex
with and affect signaling through the low affinity nerve growth factor
receptor (46), another member of the TNF receptor superfamily (47).
In addition to a protein kinase catalytic domain, mPLK/IRAK also
contains an amino-terminal domain that resembles the death domain of
proteins linked to TNF RI signaling (35). Indeed, endogenous mPLK and
TNF RI proteins can be found complexed. Moreover, overexpression of
cimPLK decreases the ability of TNF to induce the activity of
NF-
B-dependent promoters. Thus, in contrast to the
IL-1-signaling pathway, the catalytic activity of mPLK/IRAK is critical
for TNF signaling and suggests that mPLK/IRAK substrates are likely to
be components of the TNF signaling pathway.
Overexpression of catalytically inactive NIK, the protein kinase that
is believed to phosphorylate I
B kinases (14-18), blocks the
activity of mPLK/IRAK. However, overexpression of wild-type NIK in the
presence of catalytic inactive mPLK results in activation of
NF-
B-dependent promoters. These data suggest that
mPLK/IRAK is upstream of NIK and suggests a TNF signaling pathway in
which mPLK/IRAK is important for transmitting a signal from TNF RI to NIK. In support of this hypothesis, mPLK/IRAK protein can complex with
NIK. mPLK/IRAK signaling is independent of the TNF-signaling molecules
TRAF2 and TRADD, indicating that mPLK/IRAK represents a previously
undescribed TNF RI signaling pathway. The latter observation is
consistent with the analysis of TRAF2 nulligenic animals, which
revealed that TNF R1 can mediate NF-
B activation in a
TRAF2-independent manner (43, 48). These results suggest a model
whereby TNF binding to TNF RI leads to activation of mPLK/IRAK protein
kinase activity and the subsequent phosphorylation of mPLK/IRAK
substrates, leading to the activation of NF-
B.
Our data places mPLK/IRAK in the TNF RI signaling pathway and confirms
a role for mPLK/IRAK in the IL-1-signaling pathway. These data also
suggest that the regulation of mPLK/IRAK activity may be more
complicated than previously appreciated. In the IL-1 pathway, mPLK/IRAK
catalytic activity is not required and, thus, is similar to the role of
RIP in TNF signaling. Although cells lacking RIP are defective in TNF
signaling, the defect can be reversed by expression of wild-type or
catalytically inactive RIP (40, 49). How RIP or mPLK/IRAK may transduce
these effects is unclear. Our data suggests that TNF and IL-1 signaling
may be coordinated at the level of mPLK/IRAK. In response to IL-1, the
mPLK/IRAK protein is targeted for degradation (19). Interestingly, CD30
and TNF RII potentiate TNF RI-induced apoptosis by inducing the
degradation of TRAF2 (50). It thus seems possible that mediation of
cross-talk between the IL-1 and TNF RI signaling pathways may occur
through the targeted degradation of mPLK/IRAK.