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Section of Infectious Diseases, Department of Medicine, Wake Forest University School of Medicine, Winston-Salem, NC 27157
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B
activation. IRAK from leukocytes undergoes rapid activation and
inactivation/degradation following IL-1 or LPS stimulation. The rapid
degradation of IRAK may serve as a negative feedback mechanism of
down-regulating IL-1R/TLR-mediated signaling and cytokine gene
transcription. Although IL-1/IL-1R-triggered IRAK degradation has been
studied in detail, the mechanism of LPS-induced IRAK activation and
degradation is not clearly defined. In this study, we demonstrate that
the IRAK N-terminal 186-aa region is required for LPS-induced
degradation. The N-terminally truncated IRAK protein expressed in human
monocytic THP-1 cells remains stable upon LPS challenge. In
comparison, IRAK as well as the IRAK mutant with C-terminal truncation
undergo degradation with LPS stimulation. We demonstrate that
pretreatment with protein kinase C inhibitor calphostin inhibits
LPS-induced IRAK degradation. Furthermore, we observe
coimmunoprecipitation of endogenous IRAK and protein kinase C-
protein. We show that functional TLR4 is required for LPS-mediated IRAK
degradation. IRAK protein in the murine GG2EE cells harboring a mutated
TLR4 gene does not undergo degradation upon LPS treatment. In sharp
contrast, we observe that the IRAK homolog, IRAK2, does not undergo
degradation upon prolonged LPS treatment, suggesting complex regulation
of the innate immunity network upon microbial
challenge. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B activation and
trans activation of many inflammatory cytokine genes, which
regulate host innate immune response (3, 4).
IRAK also participates in Toll-like receptor (TLR)-mediated signaling.
Recent studies reveal a group of mammalian TLRs that respond to diverse
microbial stimulants including LPS and trigger NF-B activation as
well as cytokine gene transcription (5). The distinct
extracellular leucine-rich-repeat structures of various TLRs enable
them to respond to different microbial stimulants (5).
Interestingly, all TLRs share sequence similarity in their
intracellular domain with the IL-1R (Toll/IL-1R-like (TIR) domain).
Accumulating evidence indicates that IRAK is also used by the TLR
signaling in response to microbial stimulants. For example, we and
others showed that LPS can activate host innate immune response
through TLR4 and IRAK (6, 7). In addition, Gram-positive
bacterial stimulants such as lipoprotein can activate human innate
immunity through TLR2 and IRAK (8, 9). Recently, we showed
that a bacterial flagella protein FliC triggers cytokine production in
human leukocytes through TLR5 and IRAK (10). Therefore,
IRAK most likely serves as one of the central mediators of host innate
immune response upon various microbial stimulations.
Perhaps developed as a negative feedback mechanism, IRAK becomes
quickly degraded and unresponsive to further IL-1/LPS stimulation
following prolonged IL-1/LPS treatment (7, 11). The
unresponsiveness of IRAK is at least partly responsible for the
repressed cytokine gene transcription commonly seen in LPS and/or
IL-1
tolerant leukocytes (7, 12). Although most
researchers usually generate tolerance cells with overnight LPS
challenge, it is worth noting that endotoxin tolerance can be developed
as early as 2 h after pretreatment with LPS (13, 14),
coinciding with the timing of IRAK degradation. Several studies using
IL-1 as stimulant in HEK293 cells and other nonleukocyte cells
indicate that IRAK is degraded through phosphorylation-induced
ubiquitin/proteosome pathway (11, 15). Application of
kinase inhibitors or proteosome inhibitors can prevent IL-1-induced
IRAK degradation (11). Recently, two research groups
reported that two regions of IRAK are involved in IL-1-induced IRAK
degradation (aa 103–198 located between the N-terminal death domain
and the central kinase domain (16), as well as aa 514–543
at the end of the kinase domain (17)). IRAK mutant with
deletion of either region resists IL-1-induced IRAK degradation.
However, the aa 514–543 region is most likely not directly involved in
IL-1-induced IRAK phosphorylation and subsequent ubiquitination,
because mutations of any putative phosphorylation sites (Ser, Tyr, Thr)
and/or putative ubiquitination sites (Lys) within this region fail to
stabilize IRAK upon IL-1 challenge (17).
IRAK degradation upon IL-1 challenge in several nonleukocyte cell
lines (such as kidney 293 cells, fibroblast MRC-5 cells, liver HepG2
cells) has been studied in detail (11, 16, 17). However,
the biochemical mechanism of IRAK degradation and unresponsiveness in
LPS tolerant leukocytes have not been determined. The 293 cells do not
express TLR4, and are therefore not suitable for studying the effect of
LPS on IRAK stability. We chose to use the relevant human promonocytic
THP-1 cells to examine the regulation of IRAK by LPS.
Undifferentiated THP-1 cells express functional TLR4, but not type 1
IL-1R (18, 19); therefore, IL-1 stimulation has been shown
not to cause IRAK degradation in THP-1 cells (7, 20). We
can thus study specifically the effect of LPS challenge on IRAK. As
shown previously by us and others in THP-1 cells, IRAK is a
constitutively expressed protein (7, 17, 20). Upon LPS
stimulation, IRAK is quickly activated and then degraded. In the
presence of LPS, IRAK protein level constantly remains at a very low
level after 2 h of LPS stimulation (7, 17, 20). We
generated various IRAK truncation mutants and tested their stabilities
inside THP-1 cells upon 2-h LPS treatment. We observe that the
N-terminal 186 aa of IRAK is required for LPS-induced IRAK degradation.
Furthermore, we demonstrate that application of the protein kinase
(PK)C inhibitor calphostin inhibits LPS-induced IRAK degradation. In
addition, our study shows that functional TLR4 is required for
LPS-induced IRAK degradation in THP-1 cells. Interestingly, we observe
that the close homolog of IRAK, IRAK2, does not undergo degradation
upon prolonged LPS challenge in THP-1 cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CKI-7 was purchased from Seikagaku (Falmouth, MA). Calphostin and 5,6-dichloro-1-b-D-ribofuranosyl-benzimidazole were obtained from Calbiochem (La Jolla, CA). H-89 was obtained from Biomol (Plymouth Meeting, PA).
Recombinant plasmids and cell lines
The mammalian expression plasmids encoding IRAK were obtained
from X. Li (Cleveland Clinic Foundation, Cleveland, OH)
(15). A PCR fragment encoding the flag tag was inserted at
the 5' end of the IRAK coding sequence to generate pflag-IRAK. To make
the pflag-IRAK1–547, the 3' end of the IRAK
coding sequence (from base 1641 to the end) was excised out using
BsaAI and SmaI. The
pflag-IRAK186–217 was generated by substituting
the nucleotides from 1–571 with an adapter oligonucleotide. The
NF-B-luciferase reporter plasmid pNF-B-luc was purchased from
Promega (Madison, WI). Human promonocytic THP-1 cells were obtained
through American Type Culture Collection (Manassas, VA). The mouse
GG2EE and HeNC2 cells were provided by A. Ding (Cornell University
Medical College, Ithaca, NY).
THP-1 cell transfection
THP-1 cells were cultured in RPMI 1640 medium supplemented with 10% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 mM glutamine. A total of 1 x 106 cells was transfected using Effectin reagent (Qiagen, Chatsworth, CA) with 0.8 µg of the indicated IRAK expression constructs. Twenty-four hours after transfection, cells were harvested and the lyses were used in the subsequent assays.
Generation of polyclonal anti-IRAK2 Ab
A peptide TQLRKIKSMERVQG corresponding to the IRAK-2 protein aa 40–53 was synthesized and conjugated to keyhole limpet hemocyanin through our peptide synthesis core lab. Such peptide was used to immunize rabbit and generate antiserum through Pocono Rabbit Farm (Canadensis, PA).
Immunoprecipitation, Western blot, and in vitro kinase assay
THP-1 cells transiently transfected with indicated IRAK expression plasmids were harvested and lysed, as described previously (7). Cell debris was pelleted by centrifugation for 20 min at maximum speed in a microcentrifuge. The protein concentration in the supernatant was determined using a protein assay kit (Bio-Rad, Richmond, CA) according to the protocol provided by the manufacturer. Extracts with equal amount of proteins were used for the immunoprecipitation. A total of 5 µl anti-flag mAb (Sigma-Aldrich, St. Louis, MO) was added to 800 µl each of the isolated cell extracts and incubated at 4°C for 3 h on a rotator. A total of 50 µl of a 50% slurry of prewashed protein G agarose beads was then added to each sample, followed by incubation for an additional 2 h at 4°C. The samples were spun briefly in a microcentrifuge and washed for four times in lysis buffer. Samples were subsequently solubilized by SDS sample buffer (80 mM Tris-HCl (pH 6.8), 2% SDS, 50% glycerol, 0.05% bromphenol blue, 0.2 M DTT), separated by SDS-PAGE, transferred to polyvinylidene difluoride (PVDF) membrane (Bio-Rad), Western blotted with anti-flag Ab, and detected by ECL reagent.
A similar procedure, as described above, was used to prepare cell extracts from GG2EE and HeNC2 cells. Endogenous IRAK proteins from these cells were immunoprecipitated using polyclonal anti-IRAK Ab (Upstate Biotechnology, Lake Placid, NY). SDS-PAGE and Western blot analyses were performed, as described above.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Upon transient transfection of the pflag-IRAK,
pflag-IRAK186–712, or
pflag-IRAK1–547 construct into THP-1 cells, cell
lyses were prepared and the various flag-IRAK fusion proteins expressed
were immunoprecipitated using anti-flag mAb. Due to inherent low
transfection efficiency of THP-1 cells, the levels of expressed
flag-IRAK proteins are low, as shown on the gel (Fig 1
B). Two separate studies reported that low levels of
transient expressions of IRAK in EL-4 cells and HeLa cells result in a
single 80-kDa protein band (21, 22). In agreement with
such studies, we consistently observed that the full-length flag-IRAK
protein transiently expressed in THP-1 cells runs at the expected size
of 
80 kDa (Fig. 1B).
Accordingly, the truncated flag-IRAK186–712 or
flag-IRAK1–547 protein runs at the expected size
of 60 kDa (Fig. 1B).
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B). Strikingly, the
flag-IRAK186–712 with deletion of N-terminal 186
aa survives the 2-h LPS treatment (Fig. 1B). We could not
detect the phosphorylated form(s) of the expressed
flag-IRAK proteins following 2-h LPS challenge, suggesting
that these forms may be rapidly degraded once generated in THP-1 cells.
This is in agreement with two other independent studies using THP-1
cells, showing that the amount of endogenously phosphorylated IRAK
forms following prolonged LPS stimulation is very low and not readily
detectable (20, 23). Our results indicate that the
LPS-induced IRAK degradation requires the N-terminal 186 aa of IRAK,
and the C terminus region is not necessary for IRAK degradation. Our
observation agrees with the recent independent report showing that the
aa 103–198 is required for IL-1-induced IRAK
phosphorylation, ubiquitination, and degradation (16).
IRAK phosphorylation by PKC- is necessary for LPS-induced IRAK
degradation
Based on studies by us and others, it is speculated that
LPS-induced IRAK phosphorylation may facilitate its degradation through
ubiquitin pathway (11). Realizing the surmounting task of
identifying the phosphorylation sites through phospho-amino acid
sequencing analysis, we took a shortcut approach by studying the effect
of various protein kinase inhibitors on LPS-induced IRAK degradation.
THP-1 cells were pretreated for 20 min with either 1 mM CK1–7
(specific inhibitor of casein kinase-1; Seikagaku), 500 µM
5,6-dichloro-1-b-D-ribofuranosyl-benzimidazole
(specific inhibitor of casein kinase II; Calbiochem), 5 µM H-89
(specific inhibitor of PKA; Biomol), or 5 µM calphostin (specific
inhibitor of all PKC isozymes; Calbiochem). The above-mentioned
concentrations for the specified inhibitors have been shown to be
selective in inhibiting the corresponding kinases
(24, 25, 26, 27). Inhibitor-pretreated cells were subsequently
challenged with 500 ng/ml LPS for 2 h. The endogenous IRAK protein
levels were analyzed through Western blot. As shown in Fig. 2, 2-h LPS treatment renders dramatic
decrease of the IRAK protein level (Fig. 2). Pretreatments with CK-1-,
CK-II-, or PKA-specific inhibitors do not interfere with LPS-induced
IRAK degradation (Fig. 2). Strikingly, pretreatment of THP-1 cells with
general PKC inhibitor calphostin consistently renders endogenous IRAK
stable upon LPS challenge (Fig. 2). We have also tested the effect of
another general PKC inhibitor RO31-8220 on LPS-induced IRAK degradation
and obtained similar results (data not shown).
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,
, and 
isozymes), novel PKC (
, 
, 
, and 
isozymes),
and atypical PKC ( and 
isozymes) (reviewed in Ref.
28). It has been reported that LPS-treated monocytes/THP-1
cells have an elevated atypical PKC activity (29, 30).
Further study demonstrated that PKC- is specifically activated in
THP-1 cells upon LPS stimulation (29). Therefore, we have
examined whether PKC- interacts with IRAK in vivo inside THP-1
cells. Total protein extracts were prepared from LPS-treated THP-1
cells and used to immunoprecipitate IRAK protein. Immunoprecipitated
IRAK protein complex was separated on SDS-PAGE and blotted to PVDF
membrane. The membrane was probed for the presence of PKC- using
polyclonal anti-PKC- Ab. As shown in Fig. 3, we can detect the presence of PKC-
in the immunoprecipitated IRAK complex.
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Because LPS can activate leukocyte innate immune response both
extracellularly through TLR4 (31) and intracellularly
through an unknown mechanism (32), we would like to
determine whether functional TLR4 is required for LPS-induced IRAK
degradation. To test this, mouse GG2EE as well as HeNC2 macrophage
cells were used. GG2EE cell line derives from the C3H/HeJ mouse that
harbors a point mutation in TLR4 gene, rendering TLR4 unable to mediate
LPS signaling (31). HeNC2 cell line derives from the
isogenic C3H/HeN wild-type mouse strain. GG2EE as well as HeNC2 cells
with or without LPS treatment were harvested, and protein lyses were
extracted. IRAK proteins were immunoprecipitated and analyzed through
Western blot using polyclonal anti-IRAK Ab that can recognize both
the human as well as the mice form of IRAK (Upstate Biotechnology). As
shown in Fig. 4, LPS treatment causes
rapid IRAK level decrease in the wild-type HeNC2 cells. However, the
IRAK level remains constant in the GG2EE cells harboring a mutant TLR4
gene with or without LPS stimulation (Fig. 4). As a control, we treated
GG2EE and HeNC2 cells with 500 ng/ml murine IL-1 and observed that
IL-1 causes similar IRAK degradation in both cells. Our data
indicate that functional TLR4 is necessary for mediating LPS-induced
IRAK degradation.
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IRAK2 shares with IRAK sequence similarities in the N-terminal
10–100 aa death domain and the central 200–500 aa kinase domain.
However, IRAK2 bears no homology with IRAK within the 103–186 aa
region, which is required for IRAK degradation (Fig. 2). There has been
no report regarding the stability and physiological function of IRAK2
upon LPS or IL-1 challenge. We developed specific polyclonal Ab
against IRAK2 through Pocono Rabbit Farm (see Materials and
Methods) and performed Western analysis using whole protein
extracts prepared from THP-1 cells treated with or without LPS. Equal
amount of total protein extracts was separated on SDS-PAGE and blotted
with anti-IRAK2 Ab. As shown in Fig. 5, the anti-IRAK2 Ab, not
the preimmune serum, specifically detected a protein band of 60 kDa,
the expected size of IRAK2. Interestingly, we observed that the
endogenous IRAK2 level remains constant following prolonged LPS
stimulation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our results agree with the recent report, using IL-1 as stimulant in
HEK293 cells, that IRAK mutant with deletion of aa 103–198 completely
abolishes IL-1-induced IRAK phosphorylation, ubiquitination, and
degradation (16). This region resides between the
N-terminal death domain and the central kinase domain (Fig. 1). Besides
the aa 103–186 region, it is reported that aa 514–543 located at the
end of the kinase domain is also involved in IL-1-induced IRAK
degradation (17). Complete deletion of this region renders
IRAK stable and resistant to IL-1-induced modification and
degradation. However, the exact role of the aa 514–543 region is not
clear. IRAK point mutations of all putative phosphorylation sites (Ser,
Tyr, Thr) and putative ubiquitination sites (Lys) within this
region still undergo IL-1-induced phosphorylation and ubiquitination
(17). This suggests that the aa 514–543 region is not
directly involved in IRAK phosphorylation/ubiquitination. Rather, this
region might indirectly contribute to IRAK degradation, for example, by
modifying the overall secondary structure of the protein, or through
interaction with other protein(s) involved in IRAK phosphorylation and
ubiquitination.
Our work is the first to determine that functional TLR4 is necessary
for LPS-induced IRAK degradation. Previous work regarding IRAK solely
characterized the IRAK response upon IL-1/IL-1R challenge. Although
TLR4 bears similarity with IL-1R intracellular TIR domain, there
are distinct amino acid differences. In addition, unlike IL-1
signaling, which solely goes through type 1 IL-1R, LPS is known to
undergo internalization and signal through other intracellular receptor
(such as NOD-1 receptor) (32). Therefore, TLR4- or
LPS-mediated intracellular events through NOD-1 may contribute to IRAK
protein degradation. Our work using TLR4-defective murine GG2EE cells
clearly indicates that LPS-induced IRAK degradation requires
functional TLR4.
Previous study using general kinase inhibitor suggests that LPS-induced
IRAK phosphorylation contributes to its subsequent degradation
(11). Our data presented in this study further suggest
that putative IRAK phosphorylation by PKC might be responsible for its
degradation. Pretreatment of THP-1 cells with PKC inhibitor calphostin
prevents LPS-induced IRAK degradation. It is noteworthy that LPS
specifically activates PKC- isoform in THP-1 cells
(29). Correspondingly, we have documented binding of IRAK
and PKC- upon LPS stimulation. Although PKC- binding with IRAK is
intriguing, we are aware of the fact that binding alone could not prove
that IRAK has indeed been phosphorylated by PKC- in vivo. Future
extensive work using transgenic mice with PKC- deletion is needed to
test the involvement of PKC in IRAK phosphorylation and degradation.
Nevertheless, our study reported in this work provides important lead
for the elucidation of the mechanism of LPS-induced IRAK
phosphorylation/degradation.
Another interesting finding provided in this work is that the close
homolog of IRAK, IRAK2, apparently does not undergo degradation upon
LPS challenge. Homologous to IRAK, IRAK2 possesses a similar death
domain and a kinase domain. In contrast, the IRAK N-terminal 103–186
aa region required for IRAK degradation is not conserved in IRAK2 (Fig. 5). Our observation that IRAK2 is stable
indicates that the region for LPS-induced IRAK degradation resides
within the aa 100–186 region. We are the first to report in this study
that IRAK2 protein is stable upon prolonged LPS treatment. To date,
there have been very few studies regarding the regulation and
physiological function of IRAK2. A previous study using transient
expression of IRAK2 in 293 cell suggests that IRAK2 may play a
redundant role as IRAK in signaling (33). However, it was
recently reported that IRAK2 selectively binds with another adapter
protein Mal/TIRAP instead of MyD88 upon LPS stimulation, and
selectively participates in MyD88-independent dendritic cell activation
(34). This clearly indicates that IRAK2 plays distinct
roles in mediating innate immune response. Our finding that IRAK2
protein remains stable upon LPS challenge further reveals the
complexity of the innate immunity regulation upon microbial
stimulations.
Taken together, our data indicate that LPS-induced IRAK degradation requires functional TLR4. The IRAK N-terminal region is required for LPS-induced degradation. Putative IRAK phosphorylation by PKC may contribute to its subsequent degradation. Strikingly, IRAK2 protein does not undergo degradation upon prolonged LPS challenge. Our findings provide valuable basis for future analysis of the complex network of innate immunity regulation.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Liwu Li, Section of Infectious Diseases, Department of Medicine, Wake Forest University School of Medicine, Winston-Salem, NC 27157. E-mail address: lwli{at}wfubmc.edu
3 Abbreviations used in this paper: IRAK, IL-1R-associated kinase; PK, protein kinase; PVDF, polyvinylidene difluoride; TLR, Toll-like receptor; TIR, Toll/IL-1R-like.
Received for publication November 30, 2001. Accepted for publication February 13, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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