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

Abstract

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-B 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-B 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 growthinhibitory stimuli (1). Subsequent investigations revealed that MyD88 possesses a modular organization (2), with an aminoterminal 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 o...

Targeting the Toll-like Receptor/Interleukin 1 Receptor Pathway in Human Diseases: Rational Design of MyD88 Inhibitors

“…These features of the protein can be analyzed by dualtag coimmunoprecipitation assays (homodimerization and recruitment of IRAKs) and by reporter gene assays (NF-B activation). 18 By following this strategy, we were able to identify 3 residues that are required for the interaction of MyD88 with IRAKs and for MyD88-dependent activation of NF-B. 18 Remarkably, mutations in one of these residues, glutamic acid 52, was also found in patients affected by recurrent infections, 19 which thus validated our in vitro studies in a physiopathologic setting.…”

“…18 By following this strategy, we were able to identify 3 residues that are required for the interaction of MyD88 with IRAKs and for MyD88-dependent activation of NF-B. 18 Remarkably, mutations in one of these residues, glutamic acid 52, was also found in patients affected by recurrent infections, 19 which thus validated our in vitro studies in a physiopathologic setting. Our studies also revealed that the small stretch of residues in the DD of MyD88, constituted by the ␣1, ␣2, ␣3 helices and by the first portion of the ␣4 helix, offers a surface of interaction between MyD88 and IRAKs.…”

“…We proved that expression of the above-mentioned small region of the MyD88 DD as fusion protein with a green fluorescent protein (GFP) moiety interfered with TLR/IL-1R responses. 18 Thus, this short amino acidic stretch could serve as template to design decoy peptides and synthetic analogues (peptidomimetics) that might interfere with TLR/IL-1R pathways (step 3). We tested this possibility in another set of studies that targeted the TIR domain of MyD88, which also is required for homodimerization.…”

Mutational Analysis Identifies Residues Crucial for Homodimerization of Myeloid Differentiation Factor 88 (MyD88) and for Its Function in Immune Cells

“…Myc-tagged MyD88 TIR, Myc-tagged MyD88, Myc-tagged IRAK1-kinase-dead (KD) (K239S), and Myc-tagged IRAK4-KD (KK213AA), were constructed as described previously (13,27,28). All constructs for the expression of mutated MyD88 TIR or MyD88 were generated by PCR using oligonucleotides containing the mutated residues.…”

“…MyD88 has a modular structure with a death domain (DD) at the N terminus, an intermediate linker domain (ID), and a TIR domain at the C terminus (10). The DD presents a fold that resembles a Greek key bundle of six antiparallel ␣-helices (11) and allows MyD88 oligomerization and its interaction with the respective DDs of the serine-threonine kinases IRAK1/2/4, thus resulting in a multimeric complex named Myddosome (12,13). This complex propagates the signal and leads to activation of transcription factors, such as the nuclear factor B (NF-B), * This work was supported by grants from Fondazione Italiana Sclerosi Mul-the activator protein 1, and the interferon-regulatory factors, and of mitogen-activated protein kinases (MAPKs), such as the stress kinase p38 and the extracellular signal-regulated kinases 1 and 2 (ERK1/2) (14).…”