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Mechanism of dysfunction of human variants of the IRAK4 kinase and a role for its kinase activity in interleukin-1 receptor signaling.

ABSTRACT: Interleukin-1 receptor (IL1R)-associated kinase 4 (IRAK4) is a central regulator of innate immune signaling, controlling IL1R and Toll-like receptor (TLR)-mediated responses and containing both scaffolding and kinase activities. Humans deficient in IRAK4 activity have autosomal recessive primary immune deficiency (PID). Here, we characterized the molecular mechanism of dysfunction of two IRAK4 PID variants, G298D and the compound variant R12C (R12C/R391H/T458I). Using these variants and the kinase-inactive D329A variant to delineate the contributions of IRAK4's scaffolding and kinase activities to IL1R signaling, we found that the G298D variant is kinase-inactive and expressed at extremely low levels, acting functionally as a null mutation. The R12C compound variant possessed WT kinase activity, but could not interact with myeloid differentiation primary response 88 (MyD88) and IRAK1, causing impairment of IL-1-induced signaling and cytokine production. Quantitation of IL-1 signaling in IRAK4-deficient cells complemented with either WT or the R12C or D329A variant indicated that the loss of MyD88 interaction had a greater impact on IL-1-induced signaling and cytokine expression than the loss of IRAK4 kinase activity. Importantly, kinase-inactive IRAK4 exhibited a greater association with MyD88 and a weaker association with IRAK1 in IRAK4-deficient cells expressing kinase-inactive IRAK4 and in primary cells treated with a selective IRAK4 inhibitor. Loss of IRAK4 kinase activity only partially inhibited IL-1-induced cytokine and NF-?B signaling. Therefore, the IRAK4-MyD88 scaffolding function is essential for IL-1 signaling, but IRAK4 kinase activity can control IL-1 signal strength by modulating the association of IRAK4, MyD88, and IRAK1.

SUBMITTER: De S

PROVIDER: S-EPMC6166721 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

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Mechanism of dysfunction of human variants of the IRAK4 kinase and a role for its kinase activity in interleukin-1 receptor signaling.

De Saurav S Karim Fawziya F Kiessu Ezechielle E Cushing Leah L Lin Lih-Ling LL Ghandil Pegah P Hoarau Cyrille C Casanova Jean-Laurent JL Puel Anne A Rao Vikram R VR

The Journal of biological chemistry 20180816 39

Interleukin-1 receptor (IL1R)-associated kinase 4 (IRAK4) is a central regulator of innate immune signaling, controlling IL1R and Toll-like receptor (TLR)-mediated responses and containing both scaffolding and kinase activities. Humans deficient in IRAK4 activity have autosomal recessive primary immune deficiency (PID). Here, we characterized the molecular mechanism of dysfunction of two IRAK4 PID variants, G298D and the compound variant R12C (R12C/R391H/T458I). Using these variants and the kina ...[more]

PMID: 30115681

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Project description:ObjectiveRecent researches have demonstrated that inflammation-related diseases are effectively regulated by posttranslational modifications (PTMs) including phosphorylation and acetylation. Our previous study found a new acetyltransferase inhibitor, oridonin, which had a protective effect on acute liver injury (ALI). In the present study, we further investigated its protective mechanism against D-galactosamine (D-Gal) combined with lipopolysaccharide- (LPS-) induced ALI in mice.MethodsIntraperitoneal injections of LPS (40 μg/mouse)/D-Gal (5 mg/mouse) were given to the mice, and the experimental group was pretreated with intraperitoneal injection of oridonin (0.2 mg/mouse). To elucidate the protective mechanism of oridonin, we collected liver specimens and used RNA-sequencing (RNA-Seq) analysis. We focused on the genes that were upregulated by LPS/D-Gal and downregulated after pretreatment with oridonin. The downregulated genes examined by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were further verified by real-time polymerase chain reaction (PCR) and western blot.ResultsGO analysis showed that genes that were downregulated after pretreatment with oridonin were extremely concentrated in immune response, chemotaxis, and inflammatory response. Real-time PCR confirmed that the expression of these genes was upregulated by LPS/D-Gal induction and reduced after treatment with oridonin, which was consistent with RNA-Seq results. KEGG pathway analysis showed a significantly enriched downregulated gene that was present in the Toll-like receptor (TLR) 4 signaling cascade. Our results manifested that phosphorylation levels of upstream signaling molecules in the TLR4 signaling cascade, including extracellular signal-regulated kinase (ERK), P38, and IκB, were significantly inhibited by oridonin. Furthermore, LPS/D-Gal stimulation triggered posttranslational modifications of related gene loci in the TLR4 signaling pathway, including phosphorylation of IL-1 receptor-associated kinase 4 (IRAK4 T345/S346) and acetylation of IRAK4 (K34). However, after treatment with oridonin, the modification pattern of IRAK4 expression stimulated by LPS/D-Gal was suggestively attenuated.ConclusionOur study revealed that the protective effects of oridonin on LPS/D-Gal-induced ALI mediated by inhibition of the PTMs of IRAK4, including phosphorylation of T345/S346 and acetylation of K34.

Dataset Information

Mechanism of dysfunction of human variants of the IRAK4 kinase and a role for its kinase activity in interleukin-1 receptor signaling.

Publications

Mechanism of dysfunction of human variants of the IRAK4 kinase and a role for its kinase activity in interleukin-1 receptor signaling.

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