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Cited by 112NF-κB Signaling PathwaysImmune Response and InflammationCytokine Signaling Pathways and Interactions

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IRAK4 Kinase Activity Is Redundant for Interleukin-1 (IL-1) Receptor-associated Kinase Phosphorylation and IL-1 Responsiveness

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Qin, Jinzhong, Jiang, Zhengfan, Qian, Youcun, Casanova, Jean-Laurent, Li, Xiaoxia·2004·Journal of Biological Chemistry
DOI 10.1074/jbc.m400785200
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Abstract

Interleukin-1 (IL-1) stimulation leads to the recruitment of interleukin-1 receptor-associated kinase (IRAK) to the IL-1 receptor, where IRAK is phosphorylated, ubiquitinated, and eventually degraded. Kinase-inactive mutant IRAK is still phosphorylated in response to IL-1 stimulation when it is transfected into IRAK-deficient cells, suggesting that there must be an IRAK kinase in the pathway. The fact that IRAK4, another IRAK family member necessary for the IL-1 pathway, is able to phosphorylate IRAK in vitro suggests that IRAK4 might be the IRAK kinase. However, we now found that the IRAK4 kinase-inactive mutant had the same ability as the wild-type IRAK4 in restoring IL-1-mediated signaling in human IRAK4-deficient cells, including NFkappaB-dependent reporter gene expression, the activation of NFkappaB and JNK, and endogenous IL-8 gene expression. These results strongly indicate that the kinase activity of human IRAK4 is not necessary for IL-1 signaling. Furthermore, we showed that the kinase activity of IRAK4 was not necessary for IL-1-induced IRAK phosphorylation, suggesting that IRAK phosphorylation can probably be achieved either by autophosphorylation or by trans-phosphorylation through IRAK4. In support of this, only the impairment of the kinase activity of both IRAK and IRAK4 efficiently abolished the IL-1 pathway, demonstrating that the kinase activity of IRAK and IRAK4 is redundant for IL-1-mediated signaling. Moreover, consistent with the fact that IRAK4 is a necessary component of the IL-1 pathway, we found that IRAK4 was required for the efficient recruitment of IRAK to the IL-1 receptor complex.

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Crossref · 10.1074/jbc.m400785200

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Figures

Image 1
CC-BY · From the PDF
Image 2
CC-BY · From the PDF
FIG. 2 Complementation of human IRAK4-deficient cells. Human IRAK4-deficient fibroblasts were co-transfected transiently with E-selectin-luc ( A ) or AP-1-dependent luc ( B ) with increasing amounts of human wild-type, kinase-inactive, or truncated IRAK4 retroviral expression constructs. Thirty-six hours later, the cells were either untreated or stimulated for 6 h with IL-1 (100 units/ml). Luciferase activities were normalized to 𝛃 -galactosidase. Data are presented as fold induction of luciferase activity in the treated cells. The experiments were repeated four times. Shown are the data from a typical experiment. Levels of the transfected FLAG-tagged IRAK4 (IRAK4WT, IRAK4KD, and IRAK4-(1-191)) are shown by Western analysis of whole cell extracts with anti-FLAG antibody (IRAK4(anti-FLAG)), and actin was used as a loading control ( Actin ).
FIG. 2 Complementation of human IRAK4-deficient cells. Human IRAK4-deficient fibroblasts were co-transfected transiently with E-selectin-luc ( A ) or AP-1-dependent luc ( B ) with increasing amounts of human wild-type, kinase-inactive, or truncated IRAK4 retroviral expression constructs. Thirty-six hours later, the cells were either untreated or stimulated for 6 h with IL-1 (100 units/ml). Luciferase activities were normalized to 𝛃 -galactosidase. Data are presented as fold induction of luciferase activity in the treated cells. The experiments were repeated four times. Shown are the data from a typical experiment. Levels of the transfected FLAG-tagged IRAK4 (IRAK4WT, IRAK4KD, and IRAK4-(1-191)) are shown by Western analysis of whole cell extracts with anti-FLAG antibody (IRAK4(anti-FLAG)), and actin was used as a loading control ( Actin ).CC-BY · From the PDF
FIG. 3 IL-1-induced activation of NF 𝛋 B and JNK. A , NF 𝛋 B gel shift assay. Cell extracts were made from untreated or IL-1-treated (100 units/ml for 15 or 30 min) human IRAK-4-deficient fibroblasts stably transfected with empty vector, human wild-type, or kinase-inactive IRAK4. The NF 𝛋 B binding site from the IP-10 gene was used as a probe. The two bands in the gel shift assay are due mainly to p50-p65 heterodimers ( bottom ) and p65-p65 homodimers ( top ). B , JNK activation induced by IL-1. Whole cell lysates were prepared from untreated or IL-1-treated (100 units/ml for 15 or 20 min) human IRAK-4-deficient fibroblasts stably transfected with empty vector, wild-type, or kinaseinactive IRAK4 and subjected to Western blot analysis using anti-phospho-c-Jun ( P-JNK ) and anti-JNK ( JNK ) antibodies (loading control).
FIG. 3 IL-1-induced activation of NF 𝛋 B and JNK. A , NF 𝛋 B gel shift assay. Cell extracts were made from untreated or IL-1-treated (100 units/ml for 15 or 30 min) human IRAK-4-deficient fibroblasts stably transfected with empty vector, human wild-type, or kinase-inactive IRAK4. The NF 𝛋 B binding site from the IP-10 gene was used as a probe. The two bands in the gel shift assay are due mainly to p50-p65 heterodimers ( bottom ) and p65-p65 homodimers ( top ). B , JNK activation induced by IL-1. Whole cell lysates were prepared from untreated or IL-1-treated (100 units/ml for 15 or 20 min) human IRAK-4-deficient fibroblasts stably transfected with empty vector, wild-type, or kinaseinactive IRAK4 and subjected to Western blot analysis using anti-phospho-c-Jun ( P-JNK ) and anti-JNK ( JNK ) antibodies (loading control).CC-BY · From the PDF
FIG. 5 Western analysis of IL-1-induced IRAK phosphorylation and ubiquitination. Human IRAK4-deficient fibroblasts stably transfected with control vector DNA, wild-type, or kinase-inactive IRAK4 were untreated or treated with IL-1 (100 units/ml for 30 or 60 min). Cell extracts were analyzed by the Western procedure with anti-IRAK. Levels of the transfected FLAG-tagged IRAK4 (IRAK4WT and IRAK4KD) are shown by Western analysis of whole cell extracts with anti-FLAG antibody (IRAK4(anti-FLAG)), and actin was used as a loading control ( Actin ). U-IRAK , ubiquitinated IRAK. P-IRAK , phosphorylated IRAK.
FIG. 5 Western analysis of IL-1-induced IRAK phosphorylation and ubiquitination. Human IRAK4-deficient fibroblasts stably transfected with control vector DNA, wild-type, or kinase-inactive IRAK4 were untreated or treated with IL-1 (100 units/ml for 30 or 60 min). Cell extracts were analyzed by the Western procedure with anti-IRAK. Levels of the transfected FLAG-tagged IRAK4 (IRAK4WT and IRAK4KD) are shown by Western analysis of whole cell extracts with anti-FLAG antibody (IRAK4(anti-FLAG)), and actin was used as a loading control ( Actin ). U-IRAK , ubiquitinated IRAK. P-IRAK , phosphorylated IRAK.CC-BY · From the PDF
FIG. 6 Kinase-dependent E-selectin luciferase activation by IL-1 in the IRAK-deficient I1A cells. Wild-type or mutant IRAK (100 ng) and increasing amounts of kinase-inactive IRAK4 expression constructs were co-transfected with E-selectin-luc (100 ng) into the human IRAK-deficient I1A cells. The transfected cells were untreated or treated with IL-1 (100 units/ml for 6 h) followed by luciferase reporter assay. Data are presented as -fold induction of luciferase activity in the treated cells. Shown are the averages from three independent experiments. Cells transfected with vector DNA (100 ng) and E-selectin-luc DNA (100 ng) only were used as controls. Levels of the transfected FLAG-tagged IRAK4KD and IRAK (IRAKwt and IRAKmt) are shown by Western analysis of whole cell extracts with anti-FLAG antibody (IRAK4(anti-FLAG)) and anti-IRAK (IRAK), and actin was used as a loading control ( Actin ).
FIG. 6 Kinase-dependent E-selectin luciferase activation by IL-1 in the IRAK-deficient I1A cells. Wild-type or mutant IRAK (100 ng) and increasing amounts of kinase-inactive IRAK4 expression constructs were co-transfected with E-selectin-luc (100 ng) into the human IRAK-deficient I1A cells. The transfected cells were untreated or treated with IL-1 (100 units/ml for 6 h) followed by luciferase reporter assay. Data are presented as -fold induction of luciferase activity in the treated cells. Shown are the averages from three independent experiments. Cells transfected with vector DNA (100 ng) and E-selectin-luc DNA (100 ng) only were used as controls. Levels of the transfected FLAG-tagged IRAK4KD and IRAK (IRAKwt and IRAKmt) are shown by Western analysis of whole cell extracts with anti-FLAG antibody (IRAK4(anti-FLAG)) and anti-IRAK (IRAK), and actin was used as a loading control ( Actin ).CC-BY · From the PDF
FIG. 7 IL-1-induced IL-1R immune complex. A , extracts of 293-WT, I1A, I2A, or I3A, untreated or stimulated with IL-1 (100 units/ml for 10 or 20 min), were immunoprecipitated with anti-IRAK-4 followed by Western analyses with antibodies against IL-1R, MyD88, and IRAK. The expression of IRAK and MyD88 in wild-type 293 cells ( WT ) and mutant cell lines (I1A, I2A, and I3A) is shown by Western analysis of whole cell extracts ( WCE ) with anti-IRAK ( IRAK ) and anti-MyD88 ( MyD88 ). B , extracts of IRAK-4-deficient fibroblasts transfected with control vector DNA or wildtype IRAK4 were untreated or stimulated with IL-1 (100 units/ml for 30 or 60 min) and then immunoprecipitated ( IP ) with anti-IRAK followed by Western analyses with antibodies against IL-1R. The expression of IRAK4 in the transfected cells is shown by Western analysis of whole cell extracts with anti-IRAK4 ( IRAK4 ).
FIG. 7 IL-1-induced IL-1R immune complex. A , extracts of 293-WT, I1A, I2A, or I3A, untreated or stimulated with IL-1 (100 units/ml for 10 or 20 min), were immunoprecipitated with anti-IRAK-4 followed by Western analyses with antibodies against IL-1R, MyD88, and IRAK. The expression of IRAK and MyD88 in wild-type 293 cells ( WT ) and mutant cell lines (I1A, I2A, and I3A) is shown by Western analysis of whole cell extracts ( WCE ) with anti-IRAK ( IRAK ) and anti-MyD88 ( MyD88 ). B , extracts of IRAK-4-deficient fibroblasts transfected with control vector DNA or wildtype IRAK4 were untreated or stimulated with IL-1 (100 units/ml for 30 or 60 min) and then immunoprecipitated ( IP ) with anti-IRAK followed by Western analyses with antibodies against IL-1R. The expression of IRAK4 in the transfected cells is shown by Western analysis of whole cell extracts with anti-IRAK4 ( IRAK4 ).CC-BY · From the PDF

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  1. Chuang, 2000, Eur. Cytokine Netw., 11, 372
  2. Jiang, 2002, J. Biol. Chem.

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Metadata

Authors
Qin, J., Jiang, Z., Qian, Y., Casanova, J.-L., & Li, X.
Affiliations
  • Cleveland State University (US)
  • Cleveland Clinic (US)
  • Inserm (FR)
Corresponding author
Li, Xiaoxia
ORCID
Venue
Journal of Biological Chemistry
Publisher
Elsevier BV
Published
2004-06
Volume
279
Issue
25
Pages
26748-26753
ISSN
0021-9258 (ISSN-L)
DOI
10.1074/jbc.m400785200
OpenAlex
W1964985092
Cited by
110
References
27
Citations by year
2026: 3 · 2025: 2 · 2024: 1 · 2023: 3 · 2022: 5 · 2021: 3 · 2020: 5 · 2019: 2 · 2018: 8 · 2017: 2 · 2016: 4 · 2015: 1 · 2014: 6 · 2013: 2 · 2012: 1
FWCI
4.88
Type
journal article
Language
en
Open access
Licence
Locations
  • Journal of Biological Chemistry (publishedVersion, cc-by)
  • PubMed (publishedVersion)
Funders
  • National Institutes of Health
  • National Institute of General Medical Sciences (GM 60020, R01GM060020)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › NF-κB Signaling Pathways
  • Life Sciences › Immunology and Microbiology › Immunology › Immune Response and Inflammation
  • Life Sciences › Immunology and Microbiology › Immunology › Cytokine Signaling Pathways and Interactions
Keywords
IRAK4, IRAK, IL-1 signaling, IL-1 receptor, kinase redundancy, transphosphorylation, autophosphorylation, NF-κB activation, JNK activation, IL-8 expression
MeSH terms
Cell Line, Dose-Response Relationship, Drug, Enzyme Activation, Fibroblasts, Genetic Complementation Test, Humans, Interleukin-1, Luciferases, Models, Biological, Mutation, Phosphorylation, Plasmids, Precipitin Tests, Time Factors, Transfection, Blotting, Northern, Blotting, Western, Interleukin-8, NF-kappa B, Receptors, Interleukin-1, Phosphotransferases (Alcohol Group Acceptor), Genes, Reporter, E-Selectin, Mitogen-Activated Protein Kinase Kinases, Ubiquitin, JNK Mitogen-Activated Protein Kinases, MAP Kinase Kinase 4, Interleukin-1 Receptor-Associated Kinases

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