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-κB 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.
Highlights
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
Using these variants and the kinase-inactive D329A variant to delineate the contributions of Interleukin receptor–associated kinase 4 (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
Quantitation of IL-1 signaling in IRAK4-deficient cells complemented with either WT or the R12C or D329A variant indicated that the loss of myeloid differentiation primary response 88 (MyD88) interaction had a greater impact on IL-1–induced signaling and cytokine expression than the loss of IRAK4 kinase activity
Summary
To better understand the differential contributions of IRAK4 scaffolding and the kinase activity in IL-1 signaling, we characterized the expression and biochemical activities of two naturally occurring variants, G298D (4) and the compound mutant R12C/R391H/T458I (13) In both cases, the patients carry one null allele for IRAK4 and a mutated copy of the full-length protein. As one copy of IRAK4 is sufficient for normal innate immune function in humans, we initially sought to understand the molecular defects of these two variants that lead to innate immune deficiency As both proteins contain mutations in the N-terminal kinase domain, we determined whether abnormal kinase activity was the reason for the phenotype of loss of signaling. We quantified the ability of these variants to reconstitute signaling and IL-1–induced cytokine production in IRAK4-deficient cells We confirmed these findings in primary human fibroblasts using a selective IRAK4 inhibitor. We find that the IRAK4 kinase activity controls IL-1 signaling by regulating the formation and stability of the myddosome and that scaffolding function is essential for both assembly of the myddosome and IL-1 signaling
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