From the Department of Biochemistry and Molecular Biology, Indiana University School of Medicine and the Walther Cancer Institute, Indianapolis, Indiana 46202-5121
Received for publication, November 16, 2000
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ABSTRACT |
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The IL-1 receptor-associated kinase (IRAK/mPLK)
is linked to the regulation of nuclear factor- Tumor necrosis factor Inactivation of I The IL-1 receptor-associated kinase (IRAK) was identified as a protein
associated with the type I IL-1 receptor (21), whereas the mouse
homologue of IRAK, mouse pelle-like kinase (mPLK), was identified
independently (22) in a cDNA library screened for kinases related
to the Drosophila pelle kinase (23). We have demonstrated
that IRAK/mPLK protein kinase activity is critical for TNF-RI signaling
and placed mPLK/IRAK in a novel signal transduction pathway
through which TNF-RI activates NF- Library Screening--
DNA fragments encoding different regions
of the IRAK/mPLK protein (21, 24) were subcloned into pGBDU and were
transformed into the yeast strain PJ69-4A (28). Transformants were
selected on SD/ Northern Blot Analysis--
Northern blots containing
mRNA extracted from developing mouse embryos and a panel of adult
tissues (CLONTECH) were probed with a SIMPL
cDNA radiolabeled by random priming [ Plasmid Constructs and Antibodies--
The IL-8-Luc and
(NF- Cell Culture and Transfections--
Human embryonic kidney 293 (HEK 293) epithelial cells and C3H10T1/2 mouse embryo fibroblast cell
lines were maintained and transfected as described previously (24). To
monitor transfection efficiencies, precipitates also included a
construct containing the Renilla luciferase cDNA.
Cultures were harvested 24 or 48 h following transfection, and
luciferase activities were determined using the
Dual-Luciferase® reporter assay system (Promega, Madison,
WI) according to the manufacturer's specifications. Individual assays
were normalized for Renilla luciferase activity, and data
are presented as the -fold increase in activity relative to empty
vector control. Data are from 2 to 3 independent experiments performed
in duplicate or triplicate with similar qualitative results with
standard errors indicated.
TUNEL Assays--
HEK 293 cells (2 × 106) were transfected with a mammalian expression vector
encoding Immunoprecipitations and Western
Blotting--
Immunoprecipitations, SDS-polyacrylamide gel
electrophoresis, and Western blot analysis were performed as described
previously (24).
IRAK/mPLK Nulligenic ES Cells--
IRAK/mPLK nulligenic ES cells
were generated with the mPLK/IRAK-targeting construct as described by
Thomas et al. (27). The 129 Sv-derived ES cell line
R1 was maintained in Dulbecco's modified Eagle's medium supplemented
with 20% fetal bovine serum, 100 IU/ml penicillin, 100 µg/ml
streptomycin, 2 mM glutamine, 1.0 mM sodium
pyruvate, 0.1 mM 2-mercaptoethanol, and 1.2 × 103 units/ml leukemia inhibitory factor (LIF, Life
Technologies, Inc.) on monolayers of mitomycin C-treated STO cells
(subline of SIM mouse fibroblasts). To introduce the targeting
vector, 8 × 106 R1-ES cells were electroporated in
0.8 ml of cold phosphate-buffered saline containing 40 µg of the
linearized targeting construct. Electroporated cells were incubated on
ice for 20 min before co-culturing with the mitomycin-treated STO
monolayers. Three days after electroporation, cell cultures were
treated with 400 µg/ml G418 (Life Technologies, Inc.) and 2 µM gangcyclovir. Individual colonies were isolated 10 days later. Genomic DNA was purified from G418 and
gangcyclovir-resistant ES cell colonies with a DNA isolation kit
(Puregene, Minneapolis, MN). The first round of screening was performed
by polymerase chain reaction, and positives were rescreened by Southern
blot analysis. Genomic DNA was digested with the BamHI
restriction endonuclease and separated on a 0.7% agarose gel in 1× a
buffer containing 40 mM Tris acetate and 1 mM
EDTA. The DNA was transferred to a nylon membrane (MSI, Westboro, MA)
and probed with genomic fragments that distinguish the wild-type and
targeted allele using IRAK/mPLK genomic probes outside the region that
is used to generate the targeting construct. Only one targeting event
was necessary as the mPLK gene is located on the X chromosome.
Liquid Chromatography Electron-Spray Mass
Spectroscopy--
Immunocomplexed materials were subject to
SDS-polyacrylamide gel electrophoresis, and individual protein bands
were excised and digested with trypsin. Solubilized samples were
analyzed by capillary liquid chromatography using an ABI 140D solvent
delivery system. Samples were applied directly to 300-µm
inside diameter fused silica capillaries packed with Vydac C18 resin
and separated with gradients of Buffer A (2% acetonitrile and 98%
H2O containing 0.2% isopropyl alcohol, 0.1% acetic
acid, and 0.001% trifluoroacetic acid). Peptides were eluted
with a flow rate of 7 µl/min directly into the electrospray
ionization source of a Finnigan LCQ mass spectrometer. Nitrogen was
used as the sheath gas with a pressure of 35 p.s.i., and no
auxiliary gas was used. Electrospray ionization was conducted with a
spray voltage of 4.8 kV, a capillary voltage of 26 V, and a capillary
temperature of 200 °C. Spectra were scanned over a range of
200-2000 m/z. Base peak ions were trapped using a quadrupole ion trap and further analyzed with a high resolution zoom
scan using an isolation width of 3 m/z and
collision-induced dissociation scans with a collision energy of 40.
Identification of SIMPL--
The mechanism through which IRAK/mPLK
activates NF-
The endogenous pattern of SIMPL gene expression was determined by
Northern blot analysis of mRNA isolated from embryonic tissues and
a panel of adult mouse tissues. A 1.3- and 2-kb transcript hybridized to the radiolabeled SIMPL cDNA probe (Fig. 1,
B and C). The SIMPL cDNA identified in our
two-hybrid screen (1.05 kb) is most likely encoded by the 1.3-kb
transcript. Whether the 2-kb transcript corresponds to a splice variant
of SIMPL encoding a related protein or an unrelated cross-reacting
mRNA has not been determined. SIMPL mRNA levels peak in the day
10 mouse embryo and steadily decline thereafter. In adult tissues, the
highest levels of SIMPL expression is detected in the testis, brain,
kidney, liver, and heart. Upon longer exposure SIMPL transcripts are
detected in the lung and skeletal muscle.
SIMPL Interacts with IRAK/mPLK in Intact Cells--
To
determine whether an interaction between IRAK/mPLK and SIMPL can be
detected in mammalian cells, immunocomplexing assays were performed.
Antiserum to endogenous IRAK/mPLK protein was used to generate
immunocomplexes from HEK 293 cells. As a negative control,
immunocomplexes were also generated with a nonspecific mouse IgG.
Immunoprecipitated proteins were separated by SDS-polyacrylamide gel
electrophoresis, and Western blots were prepared and probed with
antiserum to IRAK/mPLK or antiserum to SIMPL. SIMPL protein is detected
in immunocomplexes generated with the IRAK/mPLK antisera (Fig.
1D, lane under Transactivation of NF-
With the goal of generating a SIMPL mutant that could be used to study
the role of SIMPL in IRAK/mPLK-dependent signaling, a SIMPL
mutant (
To define the functional relationship between IRAK/mPLK and SIMPL, we
examined whether the ability of SIMPL to induce NF-
Our group has identified a requirement for IRAK/mPLK catalytic activity
in TNF-RI-dependent induction of NF- IKK
To evaluate the role of the SIMPL protein in physical interactions
between IRAK/mPLK and the IKKs, we first examined whether expression of
a SIMPL antisense construct would decrease steady-state levels of SIMPL
protein and protein activity. Mouse embryo fibroblasts were transfected
with the IL-8 luciferase and Renilla luciferase reporter
constructs, a mammalian expression vector encoding an epitope-tagged
version of SIMPL (SIMPL-FLAG) and an increasing amount of a
SIMPL antisense construct. Because the dominant-negative allele of
SIMPL induced an apoptotic response, a mammalian expression vector
encoding CrmA was included with the transfected DNAs. In the presence
of the SIMPL antisense construct there is a dose-dependent decrease in SIMPL-induced NF-
The data presented thus far demonstrate that the SIMPL and IKK protein
activities are interdependent. Consequently, we were interested in
determining whether the IRAK/mPLK and SIMPL proteins could be found in
IKK-containing complexes.
We first examined whether IRAK/mPLK-SIMPL could be found in IKK
To determine whether there is a requirement for SIMPL in
IRAK/mPLK·IKK
In summary, SIMPL is a novel component of the
IRAK/mPLK-dependent TNF-RI signaling pathway that leads to
the activation of NF-
Several groups of investigators including ourselves have demonstrated
that mPLK/IRAK catalytic activity is not required for IL-1 induction of
an NF-
The precise function of SIMPL is unclear. SIMPL appears to facilitate
and/or regulate complex formation between IRAK/mPLK- and IKK-containing
complexes. The evidence presented herein support the existence of a
TNF-RI-dependent
IRAK/mPLK-SIMPL-IKKB
(NF-
B)-dependent gene expression. Here we describe a
novel binding partner of IRAK/mPLK that we term SIMPL
(signaling molecule that associates with the
mouse pelle-like kinase). Overexpression of
SIMPL leads to the activation of NF-
B-dependent
promoters, and inactivation of SIMPL inhibits IRAK/mPLK as
well as tumor necrosis factor receptor type I-induced NF-
B activity. Dominant inhibitory alleles of I
B kinase
(IKK
or IKK
) block the activation of NF-
B by IRAK/mPLK and
SIMPL. Furthermore, SIMPL binds IRAK/mPLK and the IKKs in
vitro and in vivo. In the presence of antisense
mRNA to SIMPL, the physical association between IRAK/mPLK and
IKK
but not IRAK/mPLK and IKK
is greatly diminished. Moreover,
dominant-negative SIMPL blocks IKK
- or IKK
-induced NF-
B
activity. These results lead us to propose a model in which SIMPL
functions to regulate NF-
B activity by linking IRAK/mPLK to
IKK
/
-containing complexes.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
(TNF
)1 is a
pro-inflammatory cytokine that functions as an activator of the innate
immune response. Produced mainly by activated macrophages, TNF
can
influence cell survival or cell death (for review see Ref. 1).
TNF
-mediated activation of nuclear factor-
B
(NF-
B)-dependent signaling is postulated to occur
exclusively through the TNF receptor type I (TNF-RI). NF-
B binds
regulatory elements in the promoters of genes that encode mediators of
the acute and chronic inflammatory response (for review see Refs.
2-4). Prototypical NF-
B is a heterodimer composed of a 50-kDa
subunit (NF-
B/p50) and a 65-kDa subunit (RelA/p65). NF-
B activity
is most likely regulated at two levels, cytoplasmic sequestration and
direct removal from DNA. An additional protein family, inhibitors of
B (I
B), is involved in both regulatory processes (for review see
Refs. 5 and 6).
B protein activity is the result of rapid
stimulus-dependent phosphorylation on two critical
amino-terminal serine residues (7-10). The phosphorylations are a
prerequisite for ubiquitin-targeted proteasome-dependent
degradation of I
B. A large multiprotein complex that includes at
least the I
B kinases (IKK
and IKK
) and the scaffolding protein
IKK
(NEMO) has been described previously (11-18). IKK
and IKK
are responsible for I
B phosphorylation and are themselves substrates
of phosphorylation events necessary for IKK activation (for review see
Refs. 19 and 20).
B-dependent gene
expression (24). Although the IRAK/mPLK protein may be required for
IL-1-induced NF-
B activity, IRAK/mPLK catalytic activity is not
required for IL-1-dependent signaling (24-26). In
mPLK/IRAK null fibroblasts IL-1 as well as in TNF-induced NF-
B, DNA
binding activity is significantly attenuated (27). In this report we
describe a novel signaling molecule that
associates with the mouse pelle-like kinase
(SIMPL) and demonstrate that SIMPL is required for
TNF-RI-dependent activation of NF-
B activity.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
Ura, SD/
Ade, SD/
His to test for autoactivation of
the reporter genes. A pGBDU construct encoding amino acids
1-533 of mPLK (pGBDU-mPLK
534-710) that did not activate
transcription in the absence of a bait plasmid was subjected to a
two-hybrid analysis. A day 17 mouse embryo Matchmaker cDNA
library (CLONTECH, Palo Alto, CA) was transformed
into PJ69-4A expressing pGBDU-mPLK
534-710. Seven million
transformants were screened for
-galactosidase activity.
Transformants were plated onto SD/
Ura/
Leu/
His, 4 mM
3-aminothiozol plates. 4-9 days later transformants were
streaked onto SD/
Ura/
Leu/
Ade plates. Replating was repeated until
all remaining colonies could grow on SD/
Ura/
Leu/
His, 4 mM 3-aminothiozol, and SD/
Ura/
Leu/
Ade plates.
-Galactosidase activation was tested by the standard
5-bromo-4-chloro-3-indolyl
-D-galactopyranoside (X-gal)
filter lift assay.
-Galactosidase-positive clones were grown on
SD/
Leu + 5-fluoro-orotic acid plates to force loss of the bait
plasmid (pGBDU-mPLK
534-710). All positive constructs were tested
for trap-independent activation by growth on SD/
Leu/
Ade and
SD/
Leu/
His, 4 mM 3-aminothiozol plates. Plasmids
purified from PF69-4A transformants were transformed into the
bacterial strains DH5
or RR1 by electroporation. Bacterial
transformants containing the pGAD10 vector constructs were selected on
M9/
Leu plates. Positives were confirmed by retransforming the
yeast strain PJ69-4A containing pGBDU-mPLK
534-710 with the positive
pGAD10 construct and selecting for growth on SD/
Ura/
Leu/
His and
SD/
Ura/
Leu/
Ade plates. Inserts were excised from the pGAD10
vector with the EcoRI restriction endonuclease and subcloned
into a mammalian expression vector (pcDNA3.0 or pcDNA3.1,
Invitrogen, Carlsbad, CA). Nucleotide sequence analysis was determined
on both strands by the Indiana University Biotechnology Facility with
an Applied Biosystems Inc. automated sequencer.
-32P]dCTP
(3000 mCi/ml) (Amersham Pharmacia Biotech) according to the
manufacturer's recommendations.
B)3-Luc reporter constructs and IRAK/mPLK expression
constructs have been described previously (24). The SIMPL cDNA was
subcloned into a mammalian expression vector that placed the SIMPL
coding region under the control of the cytomegalovirus immediate early
gene promoter (pFLAG-CMV2, Eastman Kodak). The wild-type and
catalytically inactive versions of IKK
and IKK
(15) were kindly
provided by Michael Karin (University of California, San Diego). The
mouse c-Myc monoclonal antibody 9E10 was purchased from Roche Molecular
Biochemicals. Chromatographically purified mouse IgG was purchased from
Zymed Laboratories, Inc. (South San Francisco, CA).
IRAK/mPLK, IKK
, and IKK
antisera were purchased from Santa Cruz
Biotechnology (Santa Cruz, CA). A peptide fragment corresponding to
amino acids 200-213 of SIMPL conjugated to keyhole limpet
hemocyanin was used to immunize a female chicken (Gallus Immunotech, Ontario, Canada). Crude egg lysates were enriched for
IgY-containing protein by affinity chromatography. In pilot studies, we
determined that the SIMPL antiserum could be used for Western blot
analysis but was not suitable for immunocomplexing assays (data
not shown).
SIMPL or
SIMPL plus a mammalian expression vector
encoding CrmA. To control for the presence of nonapoptotic cells, one
set of cultures was transfected with empty vector. 24 h
later cells were harvested, fixed, and permeabilized as described below. As a positive control, fixed permeabilized cells were treated with DNase I prior to analysis. To detect double strand DNA breaks, terminal deoxynucleotidyltransferase was used to add
fluorescein-labeled nucleotides to free 3' breaks in the DNA strands
with an in situ cell death detection kit (Roche Molecular
Biochemicals). Flow cytometry was used to detect cells containing
fluorescein-labeled DNA.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
B-dependent gene transcription is not
completely understood. To identify regulators and/or substrates of
IRAK/mPLK, the yeast two-hybrid system was used to screen for binding
proteins (see "Experimental Procedures" for details). Because a
Gal4-IRAK/mPLK fusion protein containing full-length IRAK/mPLK strongly
activated all reporter genes alone, IRAK/mPLK deletion constructs were
generated and tested for auto-activation. A Gal4-IRAK/mPLK fusion
protein containing amino acids 1-533 of IRAK/mPLK (deleting amino
acids 534-710) was used for large scale two-hybrid analysis. Seven
positive clones were identified and determined to contain an identical
1.05-kb insert encoding an open reading frame of 259 amino acids. The protein encoded by the clones identified in the two-hybrid screen is a
denoted signaling molecule that associates with mPLK (SIMPL); the
deduced amino acid sequence for SIMPL is shown in Fig.
1A. Data base searches
identified expressed sequence tags for rat and human SIMPL homologues
(Fig. 1A). Readily discernible protein motifs were not
identified in SIMPL using several different algorithms, nor were
Drosophila or Caenorhabditis elegans
homologues.
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Fig. 1.
SIMPL sequence, expression pattern, and
IRAK/mPLK binding activity. A, nucleotide and amino
acid sequence of SIMPL (GenBankTM accession number
AF093135). Underscored amino acid residues correspond to
those found in the SIMPL peptide identified by mass spectroscopy.
Northern blots containing mouse embryo mRNA
(CLONTECH) (B) or a panel of adult mouse
tissues (C) were probed with a SIMPL cDNA as described
under "Experimental Procedures." The faster migrating actin
transcript detected in the heart and skeletal muscle
lanes is the muscle-specific actin isoform. D, cell
lysates were prepared from HEK 293 cells, and immunoprecipitates were
generated with antiserum to IRAK/mPLK (lane under
IRAK) and analyzed by Western blot analysis with
antiserum to IRAK/mPLK (upper panel) or antiserum to SIMPL
(lower panel). An unrelated mouse IgG was used as a negative
control (lane under IgG). Molecular size markers are
indicated in kDa on the left side of the each
panel, and bold arrows indicate the location of
detected proteins.
IRAK). Neither
IRAK/mPLK nor SIMPL is found in immunocomplexes generated with an
unrelated mouse IgG (Fig. 1D, lane under IgG).
Detection of IRAK/mPLK in immunocomplexes with SIMPL suggests that the
interaction initially detected in the yeast two-hybrid screen can be
detected in intact cells. In an independent experiment, a SIMPL-derived
peptide was identified (Fig. 1A, underscored
residues) in Myc-tagged IRAK/mPLK immunocomplexes isolated from
fibroblasts that were subjected to liquid chromatography electron
spray-mass spectroscopy (see "Experimental Procedures" for
details). Thus, the interaction between IRAK/mPLK and endogenous SIMPL
can be detected using two independent analytical approaches.
B-dependent Gene
Expression by SIMPL--
NF-
B is a key regulator of the immune and
stress responses in mammals, and NF-
B activity is increased in
response to a variety of stimuli (for review see Ref. 1). Thus, we
examined whether SIMPL, like IRAK/mPLK, induces NF-
B activity (24).
In these experiments, two different reporter constructs were used: a
luciferase cDNA under the control of NF-
B-dependent
IL-8 gene promoter (IL-8-Luc) and a luciferase cDNA under the
control of three tandem NF-
B sites
((NF-
B)3-Luc). Transient transfection of a mouse
embryo fibroblast cell line with a SIMPL cDNA results in a
dose-dependent induction of IL-8-Luc and
(NF-
B)3-Luc activity (Fig.
2, A and B) with no
effect on an activating protein-1-dependent
reporter construct (data not presented). Therefore, like IRAK/mPLK,
SIMPL lies in a signaling pathway upstream of NF-
B.
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Fig. 2.
SIMPL-induced NF- B
activity. Mouse embryo fibroblasts were transiently transfected
with an IL-8-Luc reporter construct (A) or a
(NF-
B)3-Luc reporter construct (B) and
constructs encoding the Renilla luciferase cDNA and the
indicated amounts of an eukaryotic expression vector containing no
insert (pCMV), the same eukaryotic expression vector
containing the SIMPL cDNA and/or the
SIMPL cDNA.
C, HEK 293T cells were transfected with the indicated
constructs; 24 h later cultures were harvested, and TUNEL assays
were performed as described under "Experimental Procedures." The
y axis indicates the cell number, and the x axis
indicates the fluorescence intensity. The gray curve present
in each histogram depicts results obtained with nontransfected control
cells. D, mouse embryo fibroblasts were transiently
transfected with an IL-8-Luc reporter construct, the Renilla
luciferase cDNA, CrmA, and the indicated SIMPL constructs. For
experiments presented in A, B, and D,
the error bars are mean ± S.D. of duplicate samples.
All experiments were repeated three times.
SIMPL), in which the first 80 amino acid residues are
missing, was generated. In preliminary studies we noted that the
expression of
SIMPL decreased cell
survival.2 Thus, we examined
directly whether
SIMPL expression was pro-apoptotic. HEK cells were
transiently transfected with an empty vector or
SIMPL in the absence
or presence of CrmA, a nonspecific caspase inhibitor from the cowpox
virus (29). 24 h later, cultures were harvested and analyzed for
the presence of DNA double strand breaks (in situ cell death
detection kit, Roche Molecular Biochemicals). Fluorescence-activated
cell sorting analysis revealed that the expression of
SIMPL leads to
an increase in the number of cells containing double-stranded DNA
breaks as measured by an increase in fluorescence, which are not
detected in the presence of CrmA (Fig. 2C). Based on these
results, in subsequent experiments in which the
SIMPL mutant was
analyzed CrmA was also included. To determine whether the
SIMPL
mutant functioned as a dominant-negative, wild-type SIMPL was
coexpressed with increasing amounts of
SIMPL. Expression of
SIMPL
decreases in a dose-dependent manner, SIMPL-induced NF-
B
activity (Fig. 2D). Taken together, these results reveal that
SIMPL functions as a dominant-negative allele of SIMPL, and
SIMPL, like NF-
B, appears to be critical for cell survival.
B activity was
IRAK/mPLK-dependent. For these studies, a clone of embryonic stem cells nulligenic for mPLK (ES
/
, see "Experimental Procedures") was analyzed and compared with wild-type ES cells (ES
+/+). Overexpression of SIMPL in ES +/+ cells increases IL-8-Luc promoter activity 3-fold (Fig.
3A). In contrast,
overexpression of SIMPL in the ES
/
cells does not increase IL-8
promoter activity (Fig. 3A). Thus, the ability of SIMPL to
induce NF-
B activity is dependent upon the presence of the IRAK/mPLK
protein. We next determined whether IRAK/mPLK-induced NF-
B
activation is SIMPL-dependent by examining whether
SIMPL
blocked IRAK/mPLK-induced NF-
B activity. Coexpression of
SIMPL
with IRAK/mPLK inhibits IRAK/mPLK activity (Fig. 3B). Taken
together, these data support the hypothesis that SIMPL is a component
of an IRAK/mPLK-dependent pathway that controls NF-
B
activity.
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Fig. 3.
SIMPL and IRAK/mPLK activities are
interdependent. A, SIMPL requires IRAK/mPLK activity.
Wild-type R1 embryonic stem cells (ES +/+) or IRAK/mPLK
nulligenic embryonic stem cells (ES /
) were
cotransfected with an IL-8-Luc reporter construct, a construct encoding
the Renilla luciferase cDNA, and the indicated
constructs. 48 h later cultures were harvested, cell lysates were
prepared, and luciferase activities were measured as described under
"Experimental Procedures." B, IRAK/mPLK-induced NF-
B
activity is SIMPL-dependent. Mouse embryo fibroblasts were
cotransfected with a (NF-
B)3-Luc reporter construct, a
construct encoding the Renilla luciferase cDNA, and the
indicated constructs. Cell cultures were harvested, cell lysates were
prepared, and luciferase activities were measured as described under
"Experimental Procedures." C and
D, mouse embryo fibroblasts were cotransfected
with an IL-8-Luc reporter construct, a construct encoding the
Renilla luciferase cDNA, and the indicated constructs.
Cell cultures were treated with either recombinant human TNF
(rhTNF
) (100 units, Sigma) (C) or recombinant
human IL-1
(IL-1) (3000 units, a kind gift from
Hoffman-LaRoche) (D) 6 h prior to harvest. Cell lysates
were prepared, and luciferase activities were measured as described
under "Experimental Procedures." Error bars are
mean ± S.D. of duplicate samples. All experiments were repeated
three times.
B activity that occurs in a TRADD-independent manner (24). Therefore, it was of
great interest to determine whether SIMPL, like IRAK/mPLK, is required
for TNF-RI-induced NF-
B activity. Consistent with the hypothesis
that SIMPL is downstream of IRAK/mPLK,
SIMPL inhibits TNF-RI-induced
NF-
B activity (Fig. 3C). To determine whether
SIMPL-induced inhibition is specific for TNF-RI, the effect of
SIMPL on IL-1-induced NF-
B activity was measured. Expression of
SIMPL does not inhibit IL-1-induced NF-
B activity (Fig.
3D). In parallel to the effect seen when catalytically
inactive IRAK/mPLK is expressed (24-26),
SIMPL appears to enhance
IL-1-dependent induction of NF-
B activity. Thus, like
IRAK/mPLK catalytic activity, SIMPL is also required for
TNF-RI-dependent induction of NF-
B activity.
and IKK
Mutants Inhibit IRAK/mPLK and SIMPL-induced
NF-
B Activation--
Current models predict that IKK
and IKK
are required for the activation of NF-
B-dependent gene
expression (30-33). Thus, we examined whether SIMPL-induced NF-
B
activation requires IKK
and/or IKK
activity. Substitution of the
lysine residue at position 44 for methionine within IKK
(IKK
KM)
and for alanine within IKK
(IKK
KA) results in the loss of IKK
catalytic activity (15). IKK
KM or IKK
KA attenuate
IRAK/mPLK-induced NF-
B activation and SIMPL-induced NF-
B
activation (Fig. 4, A and
B, respectively). Thus, like TNF-RI (30-33), IKK
and
IKK
are necessary components of the pathway through which IRAK/mPLK
and SIMPL signal for activation of NF-
B. We next examined whether
IKK-induced NF-
B activity is affected by
SIMPL expression.
SIMPL inhibited IKK
- and IKK
-induced NF-
B activity (Fig.
4C). These data suggest a model in which SIMPL integrates
the activities of upstream activators, like IRAK/mPLK, with the
IKK-containing complex(es).
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Fig. 4.
SIMPL and
IKK /IKK
activities
are interdependent. A-C, mouse embryo fibroblasts were
cotransfected with an IL-8-Luc reporter construct, a construct encoding
the Renilla luciferase cDNA, and the indicated
constructs. Cell lysates were prepared, and luciferase activities were
measured as described under "Experimental Procedures." Error
bars are mean ± S.D. of duplicate samples. All experiments
were repeated three times.
B activity (Fig.
5A) and a decrease in the
steady-state levels of SIMPL protein (Fig. 5B). We also examined whether transfection with the SIMPL antisense construct would
result in a decrease in the steady-state level of endogenous SIMPL
protein. Consistent with the results obtained with the FLAG-tagged SIMPL construct, transfection of the SIMPL antisense construct leads to
a decrease in the steady-state level of endogenous SIMPL protein (Fig.
5C). Thus, the transfection of HEK cells with the SIMPL
antisense construct results in decreased SIMPL protein and protein
activity.
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Fig. 5.
IRAK/mPLK, SIMPL, and IKK complex
formation. A and B, HEK 293 cells were
cotransfected with an IL-8-Luc reporter construct, a construct encoding
the Renilla luciferase cDNA, a mammalian expression
vector encoding CrmA, and the indicated constructs. The antisense SIMPL
(asSIMPL) construct contains the SIMPL cDNA in reverse
orientation under the control of the cytomegalovirus immediate early
gene promoter. 24 h later cell lysates were prepared. In
A, luciferase activities were measured as described under
"Experimental Procedures." Error bars are mean ± S.D. of duplicate samples. In B, lysates were used to
generate Western blots, which were probed with antibody to the SIMPL
protein. C, HEK 293 cells were transfected with a mammalian
expression vector encoding CrmA and either an empty mammalian
expression vector ( lane) or a mammalian expression vector
encoding an antisense SIMPL construct (+lane). 24 h
later, cell cultures were harvested, lysates were prepared, and Western
blots were generated and probed with antibody to the SIMPL protein.
D, HEK 293 cells were transfected with a mammalian
expression vector encoding CrmA plus either an empty mammalian
expression vector (
lane under
IRAK) or a
mammalian expression vector encoding an antisense SIMPL construct
(+lane under
IRAK). 24 h later, HEK 293 cells were harvested, cell lysates were prepared, and immunocomplexing
assays were performed with a mouse IgG control (lane
under IgG) or an antibody to IRAK/mPLK (lanes under
IRAK). Western blots were prepared and probed with
antibody to mPLK/IRAK, SIMPL, IKK
, and IKK
. Bold
arrows indicate the location of detected proteins.
-
and/or IKK
-containing complexes. To test this hypothesis, IRAK/mPLK
antiserum or a mouse IgG control was used to generate immunocomplexes that were subjected to SDS-polyacrylamide gel electrophoresis followed by Western blotting. Analysis of the Western
blot probed with the SIMPL antiserum revealed the presence of
SIMPL in complexes obtained with the IRAK antiserum (Fig.
5D, middle lane) but not in complexes generated
with a mouse IgG control (Fig. 5D, first lane).
When the Western blot containing the immunocomplexes that were
generated with the IRAK/mPLK antiserum was probed with antisera
to IKK
or IKK
, both proteins were detected (Fig. 5D, middle lane). Based on these data, we hypothesize that
complexes containing IRAK/mPLK, SIMPL, IKK
, and IKK
can be
isolated from cells under steady-state conditions.
/IKK
complex formation, we examined
whether the SIMPL antisense construct would affect
IRAK/mPLK·IKK
/IKK
complex formation. In these experiments the
SIMPL antisense construct was introduced into HEK 293 cells, and
immunocomplexes were generated with antibody to endogenous IRAK/mPLK
protein. Western blots were prepared and probed with SIMPL, IRAK/mPLK,
IKK
, and IKK
antisera. In the IRAK/mPLK-containing
immunocomplexes isolated from cultures expressing the antisense SIMPL
construct, neither the SIMPL protein nor the IKK
proteins were
detected in association with IRAK/mPLK (Fig. 5D, last
lane). Intriguingly, the IKK
protein was detected in the
IRAK/mPLK protein complexes independent of SIMPL.
B. Several different sets of data support a
link between SIMPL and TNF-RI-dependent activation of
NF-
B. RelA
/
animals die of massive liver apoptosis on or about
15-16 days of embryogenesis (34), a defect that does not occur in RelA
/
TNF
/
animals (35). Consistent with these data, SIMPL
transcripts are found in the liver. Overexpression of wild-type SIMPL
leads to induction of the NF-
B activity, which is associated with
cell survival, and high levels of SIMPL transcripts are detected in the
brain and testis, immune-privileged tissues in which cell survival is paramount. Recently, Pfeuffer et al. (36) isolated a
truncated version of SIMPL (amino acids 52-259) in a two-hybrid screen
for proteins that bind ActA, a critical factor in the pathogenesis of a
Listeria monocytogenes infection. Intriguingly, unlike
wild-type animals, a L. monocytogenes infection in TNF
/
or TNF-RI
/
animals is lethal (37, 38).
B-dependent response (24-26). Our group has
identified a requirement for mPLK/IRAK catalytic activity in
TNF-RI-dependent induction of NF-
B activity that occurs
in a TRADD-independent manner (24). Because dominant-negative
SIMPL blocks a TNF-RI but not an IL-1-RI response, our data support a
model in which SIMPL is a component of the
mPLK/IRAK-dependent signaling pathway that requires
IRAK/mPLK catalytic activity and lies downstream of TNF-RI.
-dependent signaling pathway. Tojima
et al. (39) recently reported that NF-
B-activating kinase
couples protein kinase C activity to IKK
/
-containing complexes.
These data combined with the results presented herein and elsewhere
(for review see Ref. 40) suggest that ligand-dependent NF-
B activation events that occur coordinate with I
B protein phosphorylation and degradation converge at the level of the IKKs.
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ACKNOWLEDGEMENTS |
---|
We thank M. Karin for supplying us with IKK constructs and J. Hawes (Indiana University School of Medicine, Indianapolis, IN) for the ionizing mass spectroscopy analysis.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant AI42798 (to M. A. H.) and National Science Foundation Grant 9728069 (to M. G. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF093135.
To whom correspondence should be addressed. Tel.: 317-274-7527;
Fax: 317-274-7592; E-mail: mharrin@iupui.edu.
Published, JBC Papers in Press, November 28, 2000, DOI 10.1074/jbc.M010399200
2 E. Vig, M. Green, Y. Liu, K.-Y. Yu, H.-J. Kwon, J. Tian, M. G. Goebl, and M. Harrington, unpublished observation.
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ABBREVIATIONS |
---|
The abbreviations used are:
TNF, tumor
necrosis factor;
NF-B, nuclear factor-
B;
I
B, inhibitor of
B;
IKK, I
B kinase;
TNF-RI, TNF receptor type I;
IRAK, IL-1
receptor-associated kinase;
mPLK, mouse pelle-like kinase;
SD, synthetic-defined media;
IL, interleukin;
SIMPL, signaling molecule
that associates with the mPLK;
Luc, luciferase;
HEK, human embryonic
kidney;
TUNEL, terminal deoxynucleotidyltransferase-mediated
dUTP-biotin end labeling of fragmented DNA;
ES, embryonic stem;
kb, kilobase(s).
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