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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.


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Separation of singly phosphorylated Ser8 IRAK-4 death domain.(a) Absorbance elution spectrum at 280 nm of IRAK-4 DD using a monoQ anion exchange column. Peaks 1 and 2 eluted at approximately 70 mM and 120 mM NaCl respectively. (b) LC/MS spectrum showing deconvoluted masses of IRAK-4 death domain corresponding to peak 1 fraction. The mass of 12782 Da is non-phosphorylated IRAK-4 death domain residues (−7) to 107. (c) LC/MS spectrum showing deconvoluted masses of IRAK-4 death domain corresponding to peak 2. The mass of 12863 Da corresponded to singly phosphorylated IRAK-4 death domain residues (−7) to 107.
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f2: Separation of singly phosphorylated Ser8 IRAK-4 death domain.(a) Absorbance elution spectrum at 280 nm of IRAK-4 DD using a monoQ anion exchange column. Peaks 1 and 2 eluted at approximately 70 mM and 120 mM NaCl respectively. (b) LC/MS spectrum showing deconvoluted masses of IRAK-4 death domain corresponding to peak 1 fraction. The mass of 12782 Da is non-phosphorylated IRAK-4 death domain residues (−7) to 107. (c) LC/MS spectrum showing deconvoluted masses of IRAK-4 death domain corresponding to peak 2. The mass of 12863 Da corresponded to singly phosphorylated IRAK-4 death domain residues (−7) to 107.

Mentions: Separation of singly phosphorylated and the non-phosphorylated IRAK-4 DD was achieved on a monoQ 5/50 anion exchange column. Proteins were eluted from the column with a linear gradient of 500 mM NaCl (Fig. 2a). Peaks 1 and 2 eluted at approximately 70 mM and 120 mM NaCl respectively. Fractions from each peaks were analysed by LC/MS. Peak 1 contained a single species with a mass of 12782 ± 1 Da corresponding to unphosphorylated IRAK-4 DD (Fig. 2b). Peak 2 was also a single species but with a mass of 12863 ± 1 Da (Fig. 2c) corresponding to a singly phosphorylated IRAK-4 DD, identified as located at Serine 8.


The N-terminal loop of IRAK-4 death domain regulates ordered assembly of the Myddosome signalling scaffold
Separation of singly phosphorylated Ser8 IRAK-4 death domain.(a) Absorbance elution spectrum at 280 nm of IRAK-4 DD using a monoQ anion exchange column. Peaks 1 and 2 eluted at approximately 70 mM and 120 mM NaCl respectively. (b) LC/MS spectrum showing deconvoluted masses of IRAK-4 death domain corresponding to peak 1 fraction. The mass of 12782 Da is non-phosphorylated IRAK-4 death domain residues (−7) to 107. (c) LC/MS spectrum showing deconvoluted masses of IRAK-4 death domain corresponding to peak 2. The mass of 12863 Da corresponded to singly phosphorylated IRAK-4 death domain residues (−7) to 107.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5120336&req=5

f2: Separation of singly phosphorylated Ser8 IRAK-4 death domain.(a) Absorbance elution spectrum at 280 nm of IRAK-4 DD using a monoQ anion exchange column. Peaks 1 and 2 eluted at approximately 70 mM and 120 mM NaCl respectively. (b) LC/MS spectrum showing deconvoluted masses of IRAK-4 death domain corresponding to peak 1 fraction. The mass of 12782 Da is non-phosphorylated IRAK-4 death domain residues (−7) to 107. (c) LC/MS spectrum showing deconvoluted masses of IRAK-4 death domain corresponding to peak 2. The mass of 12863 Da corresponded to singly phosphorylated IRAK-4 death domain residues (−7) to 107.
Mentions: Separation of singly phosphorylated and the non-phosphorylated IRAK-4 DD was achieved on a monoQ 5/50 anion exchange column. Proteins were eluted from the column with a linear gradient of 500 mM NaCl (Fig. 2a). Peaks 1 and 2 eluted at approximately 70 mM and 120 mM NaCl respectively. Fractions from each peaks were analysed by LC/MS. Peak 1 contained a single species with a mass of 12782 ± 1 Da corresponding to unphosphorylated IRAK-4 DD (Fig. 2b). Peak 2 was also a single species but with a mass of 12863 ± 1 Da (Fig. 2c) corresponding to a singly phosphorylated IRAK-4 DD, identified as located at Serine 8.

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 6–8 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.


Related in: MedlinePlus