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-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.

Kinase inhibition in autoimmunity and inflammation

“…Receptor engagement has been shown to induce K63-linked polyUb chains on several subunits of the Myddosome, suggesting that this type of modification may be of particular importance in this pathway 92 . Because IRAK4 provides structural integrity to the Myddosome, it is possible that IRAK4 kinase activity-dependent and activity-independent mechanisms work in parallel to facilitate cytokine production downstream of the Myddosome 76 , 93 , 94 . Therefore, further characterization of post-translational modifications of IRAK4 may yield kinase-independent and cell-specific mechanisms of signalling.…”

Understanding early TLR signaling through the Myddosome

“…However, all groups found only a modest defect in the degradation of IκBα, and although reduced, NF‐κB activation was still observable . More recently, using highly specific IRAK4 inhibitors 2 independent groups also showed that NF‐κB and MAPK signaling was mostly intact following TLR1/2, TLR4, and IL‐1R activation in the absence of IRAK4 kinase activity in human and mouse myeloid cells . Indeed, an alternative MAPK kinase kinase 3 (MEKK3)‐dependent (TAK1‐independent) pathway has been shown to elicit NF‐κB responses in the absence of IRAK4 kinase activity downstream of TLR activation .…”

“…Importantly, a patient with an arginine to cysteine substitution at position 12 (R12C) of IRAK4 suffered from recurrent pyogenic bacterial infections similar to the IRAK4‐deficient patients . The R12C mutation has also further been shown to disrupt IRAK4 interacting with MyD88 in vitro …”

Dimethyl Fumarate Disrupts Human Innate Immune Signaling by Targeting the IRAK4–MyD88 Complex

“…Among the .4000 total cysteines quantified by isoTOP-ABPP in Cal-1 cells, cysteine 13 (C13) of IL-1R-associated kinase IRAK4 stood out as being among the most sensitive to DMF. Noting that C13 resides proximal to the interface involved in MyD88 binding (25) and adjacent to a site of mutation in IL-1R-associated kinase (IRAK) 4 (R12C) implicated in human immunodeficiency 26, we proceeded to show that DMF disrupts both IRAK4-MyD88 interactions and IRAK4-mediated TNF-a production in a C13dependent manner. Taken together, our data indicate that DMF modifies C13 of IRAK4 in human pDCs, and this reaction disrupts MyD88 binding and IRAK4 function.…”