Myd88: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References
Gene name - Myd88

Synonyms - DmMyd88, Krapfen

Cytological map position - 45C4

Function - signaling

Keywords - Dorsal group, immune response

Symbol - Myd88

FlyBase ID: FBgn0033402

Genetic map position - 2R

Classification - death domain, Toll/Interleukin receptor TIR domain

Cellular location - cytoplasmic



NCBI links: Precomputed BLAST | Entrez Gene | UniGene
BIOLOGICAL OVERVIEW

Mammalian MyD88 is an adapter protein in the signal transduction pathway mediated by interleukin-1 (IL-1) and Toll-like receptors. In Drosophila, the Toll pathway was originally characterized for its role in the dorsoventral patterning of the embryo. Like Toll, Drosophila Myd88 messenger RNA is maternally supplied to the embryo. Homozygous mutant Myd88 female flies lay dorsalized embryos that are rescued by expression of a transgenic Myd88 complementary DNA. The Drosophila Myd88 mutation blocks the ventralizing activity of a gain-of-function Toll mutation. These results show that Drosophila Myd88 encodes an essential component of the Toll pathway in dorsoventral pattern formation (Kambris, 2003).

A second study also established a role for Myd88 in dorso-ventral patterning. Myd88 was revealed by a mutation in krapfen (kra) in a genetic screen for new maternal genes involved in embryonic pattern formation. The embryos laid by homozygous kra56 females fail to gastrulate properly and die as hollow tubes of dorsal cuticle. This phenotype is undistinguishable from those caused by mutations in the dorsal group of genes. Epistasis experiments have revealed that krapfen acts between Toll and Tube. A direct interaction was detected in yeast two hybrid experiments between Krapfen and Tube, presumably mediated by the death domains present in both proteins. Tube in turn interacts with its downstream effector Pelle through death domain association. It is therefore suggested that upon Toll activation, Myd88 associates with Pelle and Tube, in an heterotrimeric complex (Charatsi, 2002).

The Toll pathway was identified in Drosophila on the basis of its role in dorsoventral patterning during early embryogenesis. Genetic screens have led to the identification of maternal effect genes involved in the generation, transmission and interpretation of the signals specifying dorsoventral polarity in the embryo. Loss-of-function mutations in 11 of these genes result in a common maternal-effect lethal phenotype: fertilized eggs laid by homozygous mutant females produce embryos in which all cells adopt the cell fate normally restricted to the cells on the dorsal surface of the embryo, thus resulting in hollow tubes of dorsal cuticle. Loss-of-function mutations in the twelfth gene, cactus, result in the opposite (ventralized) phenotype. Genetic and molecular studies are consistent with a model in which a proteolytic cascade activated on the ventral side of the embryo generates an active ligand for the transmembrane receptor Toll. Activated Toll triggers phosphorylation and degradation of the inhibitor Cactus that releases the Rel transcription factor Dorsal, allowing its nuclear translocation. In the nucleus, Dorsal directs the expression of ventral-specific genes, such as twist, and represses dorsal-specific ones. Signal transduction from Toll to Cactus requires the proteins Tube and Pelle. These two proteins co-localize at the plasma membrane and interact through their death domains. Pelle is a serine/threonine kinase that can phosphorylate itself, and also Tube and Toll. How Pelle signals to Cactus is still unknown (Kambris, 2003 and references therein).

Components of this pathway between the putative Toll ligand Spätzle and Cactus also have a major role in Drosophila adults in the control of fungal and Gram-positive bacterial infections. The output of this pathway in adults is the nuclear translocation of the Rel protein Dorsal-related immunity factor (Dif), that upregulates the transcription of antimicrobial peptide genes such as Drosomycin. The discovery of the critical role of Toll in innate immunity in flies led to the identification of homologous genes in mammals that have been called Toll-like receptors (TLRs) and which have been shown to be required for the recognition of microbial ligands. TLRs and receptors of the interleukin-1 (IL-1) family interact with the protein MyD88 to activate the Rel transcription factor NF-kappaB, and MyD88 interacts with the Pelle-related kinases of the IRAK family. These interactions are mediated by homophilic associations involving two well-defined structural domains of MyD88: the carboxy-terminal Toll/IL-1 receptor (TIR) domain interacts with the cognate domains in the intracytoplasmic tails of the TLRs, and the amino-terminal death domain mediates interaction with the corresponding domain of IRAK. Sequencing of the Drosophila genome led to the identification of a molecule related to MyD88 that interacts physically with Toll and with the kinase Pelle, and that functions upstream of Tube and Pelle (Horng, 2001; Sun , 2002; Tauszig-Delamasure, 2002). Drosophila Myd88 differs from its mammalian counterpart by the presence of a 162 amino-acid C-terminal extension following the TIR domain that is encoded by a separate exon. Flies carrying a transposon inserted at the 5' end of this gene have an impaired response to infection (Tauszig-Delamasure, 2002). Another mutant allele of Myd88 has been identified that encodes a protein devoid of its C-terminal extension. Analysis of these mutant flies reveals that Myd88 encodes a component of the dorsoventral pathway in Drosophila embryos (Kambris, 2003 and references therein).

By using coimmunoprecipitation studies, a heterotrimeric association of the death domains has been found for Myd88, Tube, and the protein kinase Pelle. Site-directed mutational analyses of interaction sites defined by crystallographic studies demonstrate that Tube recruits Myd88 and Pelle into the heterotrimer by two distinct binding surfaces on the Tube death domain. Furthermore, functional assays confirm that the formation of this heterotrimer is critical for signal transduction by the Toll pathway (Sun, 2002; Kambris, 2003).

Tube is a scaffolding protein containing an N-terminal interaction motif belonging to the death domain family, as well as C-terminal Tube repeats that mediate binding to Dorsal. Pelle is a serine/threonine-specific protein kinase with a death domain N-terminal to its catalytic domain (Sun, 2002).

Although no Tube homolog has been found in mammals, four Pelle homologs, named IL-1 receptor-associated kinases (IRAKs), have been identified: IRAK1, -2, -M, and -4 . IRAKs function in signaling by a family of Toll-like receptors, as well as the IL-1 receptor (IL-1R), each of which contains a TIR domain, a conserved cytoplasmic signaling motif. An adaptor molecule, Myd88, associates with the C-terminal TIR domain of Toll-like receptors and the IL-1 receptor and with the N-terminal death domain (DD) of IRAKs (Sun, 2002).

During the past few years, genomic sequencing has allowed the identification of Drosophila genes with mammalian homologs functioning in Toll/IL-1 receptor-signaling pathways. These genes include IKK (a homolog of mammalian IKKalpha/ß), Kenny (a homolog of mammalian IKKgamma), IK2 (a homolog of mammalian TBK1/IKKepsilon), Myd88, TAK1, three TRAF loci, and ECSIT. These genes have been studied systematically by using RNA interference (RNAi). RNAi provides a ready means to inactivate a given gene or genes and has facilitated the dissection of Drosophila signaling pathways in cultured S2 cells. To search for essential components of the Toll pathway, an RNAi-based screen was performed among these potential Drosophila NF-kappaB regulators. This approach, coupled with genetic and biochemical analyses, has allowed the dissection of the molecular interactions among death domain-containing proteins in the Drosophila Toll pathway (Sun, 2002).

To investigate the mechanism of Toll signaling, a reporter assay was used in conjunction with RNAi in cultured Drosophila cells. A constitutively active form of Toll, TollDeltaLRR, was stably expressed in S2 cells under the control of a metallothionein promoter, such that the addition of CuSO4 to the cell culture medium initiates Toll signal transduction. To assay signal transduction downstream of Toll, these S2 cells were transiently transfected with a Drosomycin-luciferase construct. Expression of TollDeltaLRR consistently induces a significant activation (~100 fold) of the Drosomycin reporter (Sun, 2002).

To confirm the efficacy of RNAi in these cells, dsRNA was generated for several genes known to function in the Drosophila Toll pathway. RNAi against Pelle, Tube, or Dorsal significantly inhibits the activation of the Drosomycin reporter, with the effect of Dorsal RNAi relatively stronger than that of Pelle or Tube RNAi. In contrast, RNAi against Cactus dramatically enhances the activation of the Toll pathway. These observations are consistent with the fact that Pelle, Tube, and Dorsal promote Toll signaling, whereas Cactus plays an inhibitory role in the pathway. In this and all subsequent experiments, Easter RNAi serves as a negative control for any nonspecific effect of dsRNA, because Easter acts upstream, and not downstream, of Toll (Sun, 2002).

Next, RNAi-based screening was performed against fly counterparts of mammalian Toll and tumor necrosis factor pathway components, specifically Drosophila IKK, IKKgamma (Kenny), IK2, Myd88, TAK1, ECSIT, TRAF1, TRAF2, and TRAF3. Expression of each of these genes was interrupted individually by RNAi in S2 cells and the effect on Toll signaling was assayed. RNAi was also conducted against combinations of genes, in particular IKK and Pelle;IKK and IKKgamma;IKK and IK2;TRAF1, 2, and 3. To determine whether requirements are specific to the Toll pathway, the same panel of RNAi analysis was conducted in S2 cells treated with LPS. An Attacin-luciferase reporter was used to indicate LPS-mediated activation of the response pathway for Gram-negative bacteria (Sun, 2002).

When the effects of RNAi on the Toll and LPS pathways were compared, it was found that Drosophila Myd88, like Tube and Pelle, is required for activation of the Drosomycin, but not the Attacin, reporter; Drosophila Myd88 is thus essential for Toll signaling. In contrast, a requirement for TAK1 was found only in the LPS pathway and no essential role was found for fly IK2, ECSIT, or TRAF 1, 2, and 3 in either Toll or LPS signaling. Inactivating IKK and IKKgamma affected both types of signaling, with the LPS pathway being more severely inhibited than the Toll pathway. These results are consistent with the fact that inactivating IKK in flies disrupts Toll-dependent axis formation in a small fraction of embryos, although neither IKK nor IKKgamma is strictly required for Toll signal transduction (Sun, 2002).

It is known that Tube acts downstream of Toll and upstream of Pelle in signal transduction. To place Drosophila Myd88 in this pathway precisely, the epistatic relationship was examined among Myd88, Tube, and Pelle. Expression of wild-type Myd88, which has been shown to activate the Drosomycin reporter, was induced. RNAi against Pelle, Tube, or Myd88 blocks this Myd88-induced activation. These results, as well as similar findings in adult flies (Tauszig-Delamasure, 2002), indicate that Myd88 acts either upstream of or in parallel to Tube (Sun, 2002).

To dissect the signaling hierarchy further, a constitutively active form of Tube was used. This Tube-initiated activation of the Drosomycin reporter does not require Myd88, but does require Pelle. Furthermore, Pelle-induced activation of the Drosomycin reporter is diminished only by RNAi against Pelle, but not Tube or Myd88. Thus, epistasis analysis defines a linear order of action, with Tube downstream of Myd88 and upstream of Pelle (Sun, 2002).

Myd88, Tube, and Pelle each contain a death domain, a motif known to form homotypic interactions. Tube and Pelle interact directly by means of their death domains. Furthermore, Myd88 has been found to coimmunoprecipitate with Pelle in S2 cells (Tauszig-Delamasure, 2002). It was therefore interesting to discover the role of binding interactions mediated by death domains in the hierarchy defined by epistasis analysis (Sun, 2002).

To assay the interaction of Myd88 with either Pelle or Tube, full-length Myd88, as well as the death domain of Pelle (PelleDD) and a slightly larger Tube death domain peptide (TubeDD*) were epitope tagged. Also, an antiserum was generated against Drosophila Myd88. Immunoprecipitation experiments, using the alpha-Myd88 for the precipitation step and alpha-V5 to detect the tagged peptides was carried out. In pair-wise experiments, substantial interaction was detected between Myd88 and the Tube death domain. (In addition, a reduction occurred in the abundance of a fast migrating Myd88 species, perhaps reflecting a TubeDD-mediated protection from proteolysis). In contrast, only a trace amount of PelleDD coprecipitated with Myd88 (Sun, 2002).

PelleDD, TubeDD, and Myd88 were co-expressed to assay for higher-order complexes. Under such conditions, the amount of Myd88-associated PelleDD was dramatically increased. Indeed, the relative amount of TubeDD and PelleDD coimmunoprecipitated with Myd88 was indistinguishable. It is concluded that Tube forms a stable complex with Myd88 and is also strictly required for the recruitment of Pelle into a complex with Myd88 (Sun, 2002).

Two alternative models were envisioned for the role of Tube in complex formation. In one, the interaction of Pelle and Tube is essential for Pelle to join the Myd88 complex. In the alternative model, Pelle can stably associate with Myd88, provided Myd88 is bound by Tube. To discriminate between these two models, interaction surface mutations were used in characterizing a dimer between the Tube and Pelle death domains (Sun, 2002).

The crystal structure of the complex formed by the death domains of Tube and Pelle suggests that residue E50 in Tube and R35 in Pelle form a salt bridge that is critical for dimer formation. By using an RNA injection bioassays, it has been demonstrated that mutation of residue 50 in Tube to lysine (E50K mutation) abolish Tube function in Toll signaling. It was therefore surprising to find that the E50K mutation has no discernible effect on the binding of the Tube death domain to Myd88 (Sun, 2002).

Although Tube E50K has an apparently wild-type interaction with Myd88, this mutation blocks the binding of Tube to Pelle in the coimmunoprecipitation assay. Furthermore, a mutational change in Pelle (Pelle R35E) that is predicted to reconstitute the salt bridge, fully restores the Tube-Pelle interaction, just as these compensatory mutations in Tube and Pelle together allow signaling in embryos. Thus, at least two types of death domain contacts are in the Toll signaling complex: one between Tube and Pelle that involves Tube E50 and a second between Tube and Myd88 that is E50-independent (Sun, 2002).

To determine whether binding to Tube is essential for Pelle recruitment into the Myd88 complex, advantage was taken of the compensatory mutations in Tube and Pelle. In cells coexpressing TubeDD and PelleDD, the association of PelleDD with Myd88 is greatly inhibited by either individual mutation, Pelle R35E or Tube E50K, that blocks the Tube-Pelle interaction. Remarkably, the simultaneous presence of these compensatory mutations restores the recruitment of PelleDD to the Myd88 complex. It is therefore concluded that Pelle must bind directly to Tube to join the Myd88 complex (Sun, 2002).

A model is proposed that describes the three way interaction between Pelle, Tube and Myd88. The Tube-mediated complex formation involves two distinct binding surfaces on Tube death domain, which allow simultaneous association of Myd88 and Pelle. Direct binding of Myd88 to Toll and of both Tube and Pelle to Cactus-bound Dorsal would result in a complex facilitating efficient signal transduction from Toll to Dorsal. On the basis of this model of the heterotrimeric death domain complex, it is predicted that expression of the wild-type death domain of either Tube or Pelle might disrupt formation of an endogenous trimeric complex and thereby interfere with the normal function of the Toll pathway. Moreover, distinct outcomes are expected for expression of mutant forms of the Tube and Pelle death domains. The E50K mutant of TubeDD, although incapable of interacting with Pelle, nevertheless binds to Myd88 and hence might interfere with the formation of the complex of Myd88, Tube, and Pelle. By the same logic, expressing the R35E mutant of Pelle, which cannot stably interact with Tube, and hence the trimeric complex, might not interfere with signaling (Sun, 2002).

To test these hypotheses, the effect of expressing Tube and Pelle death domains was assayed in the context of an active Toll pathway. Wild-type and E50K TubeDD each significantly block TollDeltaLRR-induced activation of the Drosomycin reporter, as does wild-type PelleDD. However, the R35E mutant of PelleDD, expressed at the same level as its wild-type counterpart, has no discernible effect on Drosomycin activation. These results thus confirm the predictions of the model for heterotrimer formation and demonstrate that formation of the trimeric Myd88, Tube, and Pelle complex is a critical step in Toll signaling (Sun, 2002).

The death domain was originally identified as a protein module transducing apoptotic signals. It has been found, for example, that death domain mediated interactions between Fas and FADD or between tumor necrosis factor receptor and TRADD provide the basis for assembling the death-inducing signaling complex. The death effector domain and caspase recruitment domain also form homotypic interactions involved in apoptotic signaling and are structurally similar to a death domain. These motifs, together with the death domain, comprise the death domain superfamily (Sun, 2002 and references therein).

Experimental data demonstrate that PelleDD and Myd88 are found in the same complex when each is physically associated with TubeDD. The association of three different death domains has also been implied by studies on the tumor necrosis factor receptor complex, in which TRADD was found to facilitate the recruitment of FADD or RIP to tumor necrosis factor receptor. This study has probed the nature of such a complex and it was found that Myd88, Pelle, and Tube form a heterotrimer, with the TubeDD interacting with Myd88 and PelleDD by distinct binding surfaces (Sun, 2002).

Recently, molecular modeling based on available structural data has suggested that the homotypic interaction among death domain superfamily modules could be multivalent. Higher-order multimers, such as a heterohexamer, can be modeled by docking the death domains of Fas and FADD together. Furthermore, the structural plasticity observed in the PelleDD:TubeDD dimerhypothetically allows it to accept a third death domain into a three-fold symmetric structure. Whether the death domains of Myd88, Tube, and Pelle can form such a structure, as opposed to a linear array, awaits biophysical characterization of this trimeric complex. It is noted, however, that no evidence has been provided for any physical interaction between Pelle and Myd88. In addition, the fact that the PelleDD R35E mutant fails to dominantly interfere with Toll signaling in a functional assay argues against the possibility of such a direct contact between Pelle and Myd88 (Sun, 2002).

Because Myd88 binds to Toll through interaction between TIR domains on both proteins, it is envisioned that Myd88 connects both Tube and Pelle to Toll. Toll-initiated aggregation of these signaling molecules could trigger Pelle activation. Such a model is consistent with epistasis analyses indicating a linear order of action for Toll, Myd88, Tube, and Pelle in primary signaling (Tauszig-Delamasure, 2002). Furthermore, because Dorsal binds directly to Pelle, Tube, and Cactus, it is conceivable that the entire signaling cassette exists, at least transiently, in a single complex. As suggested by both biochemical and biological assays, Pelle-catalyzed phosphorylation may then lead to both Dorsal nuclear transport and complex dissociation (Sun, 2002).

In mammalian signaling pathways initiated by either Toll-like receptors or IL-1 receptors, Myd88 associates with IRAK. Because this study shows that a third death domain is required to mediate the interaction between Myd88 and Pelle in Drosophila, does a parallel exist in mammals? Although no known Tube ortholog exists in mammals, multiple IRAKs are present. It is speculated, therefore, that two or more IRAKs may participate in one protein complex, with the death domain of one IRAK bridging the interaction of another with Myd88. In this way, a particular IRAK isoform might act together with Myd88 to regulate the activity of a second IRAK through the oligomerization of death domains, resulting in isoform-specific biological functions (Sun, 2002).


GENE STRUCTURE

cDNA clone length - 2401 bp

Bases in 5' UTR - 571

Exons - 5

Bases in 3' UTR - 216


PROTEIN STRUCTURE

Amino Acids - 537

Structural Domains

Drosophila Myd88 encodes a protein characterized by an NH2-terminal death domain that has 27% identity to the cognate domain of Myd88. This domain is followed by a 150-amino-acid TIR domain, which has the highest similarity with mammalian Myd88 (24% identity). In addition, the Drosophila gene contains an exon encoding a COOH-terminal extension that is absent from its mammalian counterpart and does not have similarity to known protein domains (Tauszig-Delamasure, 2002).

krapfen/Myd99 encodes the Drosophila homolog of Myd88, a predicted cytoplasmic adapter protein, operating in the mammalian IL-1 (Interleukin-1) pathway. It shows a modular organization, a death domain which is generally found in many members of the tumor necrosis factor receptor (TNF-R) superfamily, an intermediate domain, and a Toll cytoplasmic domain similar to that found in the Toll/Interleukin-1-like receptors family, the TIR domain (Charatsi, 2002).


Myd88: Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 20 December 2003

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