J Biol Chem. 2009 Oct 9;284(41):28093-103. Epub 2009 Aug 13.

Identification of critical residues of the MyD88 death domain involved in the recruitment of downstream kinases.External

Loiarro, M., Gallo, G., Fanto, N., De Santis, R., Carminati, P., Ruggiero, V., Sette, C.,
--- - Department of Public Health and Cell Biology, University of Rome Tor Vergata, 00133 Rome, Italy.
MyD88 couples the activation of the Toll-like receptors and interleukin-1 receptor superfamily with intracellular signaling pathways. Upon ligand binding, activated receptors recruit MyD88 via its Toll-interleukin-1 receptor domain. MyD88 then allows the recruitment of the interleukin-1 receptor-associated kinases (IRAKs). We performed a site-directed mutagenesis of MyD88 residues, conserved in death domains of the homologous FADD and Pelle proteins, and analyzed the effect of the mutations on MyD88 signaling. Our studies revealed that mutation of residues 52 (MyD88(E52A)) and 58 (MyD88(Y58A)) impaired recruitment of both IRAK1 and IRAK4, whereas mutation of residue 95 (MyD88(K95A)) only affected IRAK4 recruitment. Since all MyD88 mutants were defective in signaling, recruitment of both IRAKs appeared necessary for activation of the pathway. Moreover, overexpression of a green fluorescent protein (GFP)-tagged mini-MyD88 protein (GFP-MyD88-(27-72)), comprising the Glu(52) and Tyr(58) residues, interfered with recruitment of both IRAK1 and IRAK4 by MyD88 and suppressed NF-kappaB activation by the interleukin-1 receptor but not by the MyD88-independent TLR3. GFP-MyD88-(27-72) exerted its effect by titrating IRAK1 and suppressing IRAK1-dependent NF-kappaB activation. These experiments identify novel residues of MyD88 that are crucially involved in the recruitment of IRAK1 and IRAK4 and in downstream propagation of MyD88 signaling.
PMID: 19679662External
Arrow2 In vitro interaction Arrow2 In vivo interaction Arrow2 Characterization Arrow2 Functional role Arrow2 top
In vivo interaction
  Endogenous
expression
Overexpression DD1 DD2 Reference
Family DD1 DD2 Method Species Region Species Region
DD IRAK1 Link Myd88 Co-immunoprecipitation HEK293 Not specified Not specified Not specified Not specified 19679662
DD IRAK1 Link Myd88 Co-immunoprecipitation HEK293 Not specified 27-72 Not specified Not specified 19679662
DD IRAK1 Link Myd88 Co-immunoprecipitation HEK293 Not specified Not specified Not specified 27-72 19679662
DD IRAK4 Link Myd88 Co-immunoprecipitation HEK293 Not specified Not specified Not specified Not specified 19679662
DD Myd88 Link Myd88 Co-immunoprecipitation HEK293 Not specified Not specified Not specified Not specified 19679662
(Link: click this icon to show interactions only between the two corresponding DDs)
Arrow2 In vitro interaction Arrow2 In vivo interaction Arrow2 Characterization Arrow2 Functional role Arrow2 top
Characterization
  Stoichiometry Affinity Protein-Protein interface Reference
Family DD1 DD2 Method Mutation Complex structure
DD IRAK1 Link Myd88 Co-immunoprecipitation E52A and E53A, Y58A, K95A (Myd88) 19679662
DD IRAK4 Link Myd88 Co-immunoprecipitation E52A and E53A, Y58A, K95A (Myd88) 19679662
(Link: click this icon to show interactions only between the two corresponding DDs)
Arrow2 In vitro interaction Arrow2 In vivo interaction Arrow2 Characterization Arrow2 Functional role Arrow2 top
Functional Role
Family DD1 DD2 Method Death-related Death-unrelated Reference
DD IRAK1 Link Myd88 Luciferase assay NF-kB-activation 19679662
(Link: click this icon to show interactions only between the two corresponding DDs)