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The N-terminal loop of IRAK-4 death domain regulates ordered assembly of the Myddosome signalling scaffold

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ABSTRACT

Activation of Toll-like receptors induces dimerization and the recruitment of the death domain (DD) adaptor protein MyD88 into an oligomeric post receptor complex termed the Myddosome. The Myddosome is a hub for inflammatory and oncogenic signaling and has a hierarchical arrangement with 6–8 MyD88 molecules assembling with exactly 4 of IRAK-4 and 4 of IRAK-2. Here we show that a conserved motif in IRAK-4 (Ser8-X-X-X-Arg12) is autophosphorylated and that the phosphorylated DD is unable to form Myddosomes. Furthermore a mutant DD with the phospho-mimetic residue Asp at this position is impaired in both signalling and Myddosome assembly. IRAK-4 Arg12 is also essential for Myddosome assembly and signalling and we propose that phosphorylated Ser8 induces the N-terminal loop to fold into an α-helix. This conformer is stabilised by an electrostatic interaction between phospho-Ser8 and Arg12 and would destabilise a critical interface between IRAK-4 and MyD88. Interestingly IRAK-2 does not conserve this motif and has an alternative interface in the Myddosome that requires Arg67, a residue conserved in paralogues, IRAK-1 and 3(M).

No MeSH data available.


Regulatory function of the IRAK-4 N-terminal loop.(A) HEK293/TLR4-MD2-CD14 cells were transiently transfected with 10 ng or 20 ng of Myc-tagged IRAK4 constructs. Cells were harvested 24 hours post-transfection. NF-κB activation was quantified by dual luciferase assay. One representative of three independent experiments is shown. Statistical Analysis—p values were determined using multiple t test and designated with p < 0.01 (**) and p < 0.05 (*) One experiment of three biological replicates is shown. (B) Expression of all IRAK4 mutants is comparable with wild type IRAK4. HEK293/TLR4-MD2-CD14 cells were transfected with Myc-tagged IRAK4 constructs and analyzed by immunoblot (IB). One experiment of three biological replicates is shown. (C) Lumier assays: MyD88 and IRAK were tagged with renilla (Ren) or protein A (ProA) at the N- or C-terminal, as indicated. KD = kinase dead. One representative out of three identical experiments shown. **p < 0.01 measured by Student’s t-test.
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f6: Regulatory function of the IRAK-4 N-terminal loop.(A) HEK293/TLR4-MD2-CD14 cells were transiently transfected with 10 ng or 20 ng of Myc-tagged IRAK4 constructs. Cells were harvested 24 hours post-transfection. NF-κB activation was quantified by dual luciferase assay. One representative of three independent experiments is shown. Statistical Analysis—p values were determined using multiple t test and designated with p < 0.01 (**) and p < 0.05 (*) One experiment of three biological replicates is shown. (B) Expression of all IRAK4 mutants is comparable with wild type IRAK4. HEK293/TLR4-MD2-CD14 cells were transfected with Myc-tagged IRAK4 constructs and analyzed by immunoblot (IB). One experiment of three biological replicates is shown. (C) Lumier assays: MyD88 and IRAK were tagged with renilla (Ren) or protein A (ProA) at the N- or C-terminal, as indicated. KD = kinase dead. One representative out of three identical experiments shown. **p < 0.01 measured by Student’s t-test.

Mentions: The Ser8 phosphorylation site characterised above lies on the outside surface of the Myddosome helix but does not contribute to a DD-DD interface. This suggests that the inability of Ser8P IRAK-4 to form a Myddosome is an indirect effect. We hypothesised that the phosphate group causes a conformational change that pulls this residue out from the Type 2 interfaces with MyD88 (Fig. 7B) causing a destabilisation of the complex. The Ser-X-X-X-Arg motif is highly conserved in vertebrate IRAK-4s but is not present in the other three IRAK paralogues (Fig. 5A,B). It is however found in the Drosophila IRAK homologues tube and pelle. To test this idea we carried out Myddosome assembly and signalling assays using several mutant IRAK-4 death domains (Fig. 6A–C). The phosphomimetic Ser8Asp mutation significantly impaired both assembly and signalling. By contrast the Ser8Arg mutation that introduces a positive charge enhanced the formation of Myddosomes in vitro and signalling to NFκB. A number of different mutants of Arg12 were also tested and all of these showed reduced levels of NFκB activation. One of these mutants, Arg12Gln did not support Myddosome assembly, consistent with the properties of the naturally occurring mutation in IRAK-4 Arg12Cys (see below). We also tested the ability of kinase dead IRAK-4 to bind MyD88 with LUMIER protein-protein interaction assays (Fig. 6D) (see ref. 19). These experiments show that binding of the kinase dead IRAK-4 mutant to MyD88 is enhanced by about 5-fold as compared to the wild-type, consistent with the hypothesis that activation of IRAK-4 kinase regulates Myddosome assembly.


The N-terminal loop of IRAK-4 death domain regulates ordered assembly of the Myddosome signalling scaffold
Regulatory function of the IRAK-4 N-terminal loop.(A) HEK293/TLR4-MD2-CD14 cells were transiently transfected with 10 ng or 20 ng of Myc-tagged IRAK4 constructs. Cells were harvested 24 hours post-transfection. NF-κB activation was quantified by dual luciferase assay. One representative of three independent experiments is shown. Statistical Analysis—p values were determined using multiple t test and designated with p < 0.01 (**) and p < 0.05 (*) One experiment of three biological replicates is shown. (B) Expression of all IRAK4 mutants is comparable with wild type IRAK4. HEK293/TLR4-MD2-CD14 cells were transfected with Myc-tagged IRAK4 constructs and analyzed by immunoblot (IB). One experiment of three biological replicates is shown. (C) Lumier assays: MyD88 and IRAK were tagged with renilla (Ren) or protein A (ProA) at the N- or C-terminal, as indicated. KD = kinase dead. One representative out of three identical experiments shown. **p < 0.01 measured by Student’s t-test.
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f6: Regulatory function of the IRAK-4 N-terminal loop.(A) HEK293/TLR4-MD2-CD14 cells were transiently transfected with 10 ng or 20 ng of Myc-tagged IRAK4 constructs. Cells were harvested 24 hours post-transfection. NF-κB activation was quantified by dual luciferase assay. One representative of three independent experiments is shown. Statistical Analysis—p values were determined using multiple t test and designated with p < 0.01 (**) and p < 0.05 (*) One experiment of three biological replicates is shown. (B) Expression of all IRAK4 mutants is comparable with wild type IRAK4. HEK293/TLR4-MD2-CD14 cells were transfected with Myc-tagged IRAK4 constructs and analyzed by immunoblot (IB). One experiment of three biological replicates is shown. (C) Lumier assays: MyD88 and IRAK were tagged with renilla (Ren) or protein A (ProA) at the N- or C-terminal, as indicated. KD = kinase dead. One representative out of three identical experiments shown. **p < 0.01 measured by Student’s t-test.
Mentions: The Ser8 phosphorylation site characterised above lies on the outside surface of the Myddosome helix but does not contribute to a DD-DD interface. This suggests that the inability of Ser8P IRAK-4 to form a Myddosome is an indirect effect. We hypothesised that the phosphate group causes a conformational change that pulls this residue out from the Type 2 interfaces with MyD88 (Fig. 7B) causing a destabilisation of the complex. The Ser-X-X-X-Arg motif is highly conserved in vertebrate IRAK-4s but is not present in the other three IRAK paralogues (Fig. 5A,B). It is however found in the Drosophila IRAK homologues tube and pelle. To test this idea we carried out Myddosome assembly and signalling assays using several mutant IRAK-4 death domains (Fig. 6A–C). The phosphomimetic Ser8Asp mutation significantly impaired both assembly and signalling. By contrast the Ser8Arg mutation that introduces a positive charge enhanced the formation of Myddosomes in vitro and signalling to NFκB. A number of different mutants of Arg12 were also tested and all of these showed reduced levels of NFκB activation. One of these mutants, Arg12Gln did not support Myddosome assembly, consistent with the properties of the naturally occurring mutation in IRAK-4 Arg12Cys (see below). We also tested the ability of kinase dead IRAK-4 to bind MyD88 with LUMIER protein-protein interaction assays (Fig. 6D) (see ref. 19). These experiments show that binding of the kinase dead IRAK-4 mutant to MyD88 is enhanced by about 5-fold as compared to the wild-type, consistent with the hypothesis that activation of IRAK-4 kinase regulates Myddosome assembly.

View Article: PubMed Central - PubMed

ABSTRACT

Activation of Toll-like receptors induces dimerization and the recruitment of the death domain (DD) adaptor protein MyD88 into an oligomeric post receptor complex termed the Myddosome. The Myddosome is a hub for inflammatory and oncogenic signaling and has a hierarchical arrangement with 6&ndash;8 MyD88 molecules assembling with exactly 4 of IRAK-4 and 4 of IRAK-2. Here we show that a conserved motif in IRAK-4 (Ser8-X-X-X-Arg12) is autophosphorylated and that the phosphorylated DD is unable to form Myddosomes. Furthermore a mutant DD with the phospho-mimetic residue Asp at this position is impaired in both signalling and Myddosome assembly. IRAK-4 Arg12 is also essential for Myddosome assembly and signalling and we propose that phosphorylated Ser8 induces the N-terminal loop to fold into an &alpha;-helix. This conformer is stabilised by an electrostatic interaction between phospho-Ser8 and Arg12 and would destabilise a critical interface between IRAK-4 and MyD88. Interestingly IRAK-2 does not conserve this motif and has an alternative interface in the Myddosome that requires Arg67, a residue conserved in paralogues, IRAK-1 and 3(M).

No MeSH data available.