Identification of Critical Residues of the MyD88 Death Domain Involved in the Recruitment of Downstream Kinases

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 Glu⁵² and Tyr⁵⁸ residues, interfered with recruitment of both IRAK1 and IRAK4 by MyD88 and suppressed NF-κB 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-κB 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.MyD88 was first discovered during studies addressing the differentiation of mouse myeloid cells in response to growth-inhibitory stimuli (1). Subsequent investigations revealed that MyD88 possesses a modular organization (2), with an amino-terminal death domain (DD),³ found in proteins involved in cell death (3, 4), and a carboxyl-terminal Toll-interleukin-1 receptor (TIR) domain, present in the intracytoplasmic tail of receptors belonging to the Toll-like receptor (TLR)/interleukin-1 receptor (IL-1R) superfamily (5). MyD88 also has an intermediate domain (ID) that is crucial in TLR signaling due to its interaction with IRAK4 (6). The role of MyD88 as a signal transducer was first shown in the pathways triggered by the activation of IL-1R (7, 8) and TLR4 (9). Further studies showed that all TLRs, with the sole exception of TLR3, and the IL-1R family utilize the adaptor protein MyD88 to initiate their signaling pathway (10).By virtue of its modular organization, MyD88 critically bridges activated receptor complexes to downstream adaptors/effectors. Upon activation, MyD88 is recruited through its TIR domain by the homologous domain of the activated TLR/IL-1R (11, 12). MyD88, in turn, has been shown to interact with a family of downstream kinases, namely IRAK1 (13), IRAK2 (7), IRAK-M (15), and IRAK4 (16), through the interaction of its DD with the respective DDs present in the amino-terminal region of IRAKs (17). At this stage, this multimeric complex is competent to elicit the propagation of the signal downstream of the receptor(s). Although MyD88 recruits IRAK-1 via DD-DD interactions, its recruitment of IRAK-4 appears to be rather unusual. Burns et al. (6) first demonstrated that an alternatively spliced variant of MyD88 (MyD88s), lacking the ID domain, failed to interact with IRAK-4, suggesting that residues located in both the DD and ID of MyD88 are crucially involved in the recruitment of IRAK-4. Nevertheless, no information is available on the specific residues in the DD in MyD88 required for its interaction with either IRAK1 or IRAK4.The DD was initially defined as the region of homology between the cytoplasmic tails of the FAS/Apo1/CD95 and TNF receptors required for their induction of cytotoxic signaling (18, 19). In analogy with other DD-containing proteins, this domain in MyD88 is also involved in the formation of homomeric and heteromeric interactions. Herein, we have undertaken an alanine-scanning mutational analysis to identify amino acids that are required for downstream signaling and might participate in the homomeric and heteromeric interactions. Our studies revealed that MyD88_E52A and MyD88_Y58A mutants are strongly impaired in the recruitment of both IRAK1 and IRAK4, whereas the MyD88_K95A mutant is deficient in recruiting IRAK4. These findings identify residues within the DD of MyD88 crucially involved in the formation of higher order complexes containing IRAK1 and IRAK4 and required for the propagation of the TLR/IL1-R signaling pathways.