tube

Protein Interactions

The interaction of the serine/threonine kinase Pelle and adaptor protein Tube through their N-terminal death domains leads to the nuclear translocation of the transcription factor Dorsal and activation of zygotic patterning genes during Drosophila embryogenesis. Crystal structure of the Pelle and Tube death domain heterodimer reveals that the two death domains adopt a six-helix bundle fold and are arranged in an open-ended linear array with plastic interfaces mediating their interactions. The Tube death domain has an insertion between helices 2 and 3, and a C-terminal tail making significant and indispensable contacts in the heterodimer. In vivo assays of Pelle and Tube mutants have confirmed that the integrity of the major heterodimer interface is critical to the activity of these molecules (Xiao, 1999).

Dorsal is an embryonic phosphoprotein. Its phosphorylation state is regulated by an intracellular signaling pathway initiated by the transmembrane receptor Toll. Immunoblot analysis of cytoplasm from precisely staged embryos reveals that the phosphorylation state of Dorsal is altered during the time period that Toll is activated. Moreover, mutations that constitutively activate Toll stimulate Dorsal phosphorylation, while mutations that block Toll activation reduce the level of Dorsal phosphorylation. Signal-dependent Dorsal phosphorylation is modulated by three intracellular proteins: Pelle, Tube, and Cactus. Free Dorsal is a substrate for a signal-independent kinase activity. This suggests that Dorsal is a substrate for a Toll-dependent kinase. (Gillespie, 1994).

A complex signal transduction pathway functions in the early Drosophila embryo to establish dorsal-ventral polarity. Activation of this pathway results in the nuclear transport of the protein dorsal (dl), a member of the rel/NF-kappaB family of transcription factors. Genetic studies have identified three intracellular components whose activities are required for activation of dl: Toll, a transmembrane receptor; pelle (pll), a serine/threonine protein kinase, and tube, a protein of unknown function. The activities of these proteins were examined when coexpressed in Drosophila Schneider cells. Coexpression of pll with dl enhances dl nuclear localization and results in a modest increase in transcriptional activity. However, when pll is coexpressed with a specific mutant derivative of Toll (TlNaeI), although not with wild-type Toll, a striking synergistic activation of dl is detected. Unexpectedly, coexpression of pll plus TlNaeI, in the absence of dl, results in a similar synergistic activation of a GAL4-tube fusion protein. Based on these and other results, a model is proposed in which pll receives a signal from activated Toll and phosphorylates tube, which then participates directly in dl activation. The C-terminal intracytoplasmic region of Toll contains a 200 residue IL-1R (type I interleukin-1 receptor) homologous domain that is essential for Toll activity, plus an extra 68 residues of unique sequence at the very C terminus. It has been shown that eletion of this unique region increases transcriptional activity of Dorsal in cotransfected Schneider cells, suggesting it plays an inhibitory function (Norris, 1996).

Tube and Pelle are required to relay the signal from Toll to the Dorsal-Cactus complex. In a yeast two-hybrid assay, Tube and Pelle interact with Dorsal. These interactions have been confirmed in an in vitro binding assay. Tube interacts with Dorsal via its C-terminal domain, whereas full-length Pelle is required for Dorsal binding. Tube and Pelle bind Dorsal in the N-terminal domain 1 of the Dorsal Rel homology region rather than at the Cactus binding site. Domain 1 has been found to be necessary for Dorsal nuclear targeting. Genetic experiments indicate that Tube-Dorsal interaction is necessary for normal signal transduction. A model is presented in which Tube, Pelle, Cactus, and Dorsal form a multimeric complex that represents an essential aspect of signal transduction (Yang, 1997).

Within the Drosophila embryo, Tube and the protein kinase Pelle transduce an intracellular signal generated by the transmembrane receptor Toll. This signal directs import of Dorsal into ventral and ventrolateral nuclei, thereby establishing dorsoventral polarity. Tube protein associates with the plasma membrane during interphase. Tube sequences required for signaling interact with Pelle in a yeast two-hybrid assay. Fusion of the Pelle catalytic domain to the transmembrane receptor Torso is sufficient to induce ventral fates; this activity is independent of Toll or Tube. Fusion of the Tube protein to Torso also induces ventral fates, but only in the presence of functional Pelle. Thus Tube appears to activate Pelle by recruiting it to the plasma membrane, thereby propagating the axis-determining signal (Galindo, 1995).

The Tube protein also functions in a novel way to enhance DL activity. In the absence of DL, or when DL is cytoplasmic, Tube is also found in the cytoplasm. But when DL is localized to the nucleus, so is Tube. Tube then functions to enhance Dorsal reporter gene expression. Tube thus appears capable of acting both as a chaperon or escort for DL as it moves to the nucleus, and then as a transcriptional coactivator, functioning in partnership with Dorsal. The intracytoplasmic domain of Toll, and specifically the region sharing homology with the interleukin-1 receptor, is sufficient to induce DL-Tube nuclear translocation (Norris, 1995).

A signaling pathway active on the ventral side of the Drosophila embryo defines dorsoventral polarity. A cell surface signal relayed by Toll, Tube and Pelle releases the Rel-related protein Dorsal from its cytoplasmic inhibitor Cactus; free Dorsal translocates into nuclei and directs expression of ventral fates. Using the yeast two-hybrid system and immunoprecipitation experiments, scaffolding and anchoring interactions were defined among the pathway components. Dorsal binds specifically to Tube, Pelle and Cactus, and the protein kinase activity of Pelle differentially regulates its interactions with Dorsal and Tube. Amino acids 47-345 oof Dorsal are sufficient for interaction with both Tube and Pelle. This same region, the Rel homology domain, is also required for dimerization, for DNA binding and for interaction with Cactus. The Dorsal Rel domain is both necessary and sufficient for generation of a dorsoventral nuclear concentration gradient. Pelle and Dorsal interact with two separable domains of Tube. Pelle binds to the amino-terminal region of Tube that spans residues 25 to 173. Dorsal binds a C-terminal domain of Tube (amino acids 257 to 462). This region contains five copies of an evolutionarily conserved, 8-amino-acid repeat and is required for full Tube function. Interaction with Dorsal requires full-length Pelle. In contrast, only about 100 amino acids of Pelle (residues 26 to 129) are necessary and, most likely, sufficient for interaction with Tube. Pelle catalytic activity modulates its interaction with Dorsal and Tube. Drosophila Filamin (an Actin binding protein that localizes to the inner surface of the cell membrane) is identified as a potential adaptor linking the interaction network, via Tube, to the transmembrane receptor Toll. The Toll/IL-1 receptor homology appears to be both necessary and sufficient for the interaction of Toll with Filamin. The studies reported here have defined minimal interactions for Pelle (residues 26-129) and Tube (residues 25-173) that correspond closely to regions with similarity to a consensus death domain (see Reaper). Death domains have been identified in pathways regulating apoptosis, but their participation in the dorsoventral signaling cascade suggests a more general role in protein interactions mediating signal transduction (Edwards, 1997).

The Toll pathway recruits Tube and Pelle to the plasma membrane, a function that is required for the transmission of information from activated Toll receptors to the Dorsal-Cactus complex. An mRNA microinjection assay has demonstrated that targeting of either Tube or Pelle to the plasma membrane by myristylation is sufficient to activate the signal transduction pathway that leads to Dorsal nuclear translocation. Using confocal immunofluorescence microscopy it has also been shown that activated Toll induces a localized recruitment of Tube and Pelle to the plasma membrane. Together, these results strongly support the hypothesis that intracellular signaling requires the Toll-mediated formation of a membrane-associated complex containing both Tube and Pelle (Towb, 1998).

Tube and Pelle have been shown to signal constitutively when fused to the extracellular and transmembrane domains of the Drosophila Torso receptor. It has been proposed that constitutive activation of these chimeras is the direct result of membrane localization. However, the Torso sequences could also have activated the chimeras by (for example) mediating an ectopic extracellular protein-protein interaction. To determine whether membrane association is in fact sufficient to constitutively activate Tube and Pelle, Src90, the amino-terminal 90 amino acids of the Drosophila Src tyrosine kinase, was fused to full-length Tube and to the catalytic domain of Pelle. The Src 90 domain contains a myristylation signal that directs association with the plasma membrane. The src90-pelle and src90-tube fusion constructs were transcribed in vitro and then assayed their activity by microinjecting the resulting RNAs into syncytial blastoderm embryos. The ability of microinjected RNA transcripts to stimulate dorsoventral signaling was analyzed by examining cell fate markers in the cuticle secreted by the developing embryo. In wild-type embryos, Toll-mediated signaling to Dorsal leads to the formation of filzkšrper and ventral denticle belts (cuticle structures representative of dorsolateral and ventral ectodermal fates, respectively). Mutations exist in which intracellular signaling is abolished due to a block in the extracellular pathway required for Toll ligand activation. For example, in embryos generated by females lacking function at the gastrulation defective locus (gd2 embryos), Toll is inactive and Dorsal remains exclusively cytoplasmic. Such embryos fail to form filzkšrper and ventral denticles. Injection of either SRC90-TUBE or SRC90-PELLE mRNA into gd2 embryos directs signaling at the posterior site of microinjection, as evidenced by the appearance of filzkšrper. SRC90-PELLE mRNA also induces the ectopic expression of ventral denticle belts. These results demonstrate the ability of both Src90-Tube and Src90-Pelle to direct Dorsal nuclear translocation in the absence of Toll activation. It is concluded that Pelle and Tube are activated upon targeting to the plasma membrane (Towb, 1998).

In the embryo, the majority of the Tube protein molecules associates with the cell surface and this association occurs over the entire circumference of the embryo. Since this localization is not restricted to ventral regions, it cannot represent an activated state of Tube. However, based on the Src90 fusion experiments, it is envisioned that Toll activates Tube by driving association of Tube with the plasma membrane. To detect any such signal-related membrane association, a detailed examination of Tube localization was carried out, using the nuclear concentration gradient of Dorsal as a marker for the dorsoventral axis. Surface views of wild-type embryos reveal that Tube forms a mesh-like array. Such a localization pattern is characteristic of proteins associated with the membranes and cytoskeleton that cap and surround each blastoderm nucleus. For Tube, the mesh-like pattern is most intense along the ventral midline, suggesting a concentration of Tube in this region. Confocal cross-sectional views confirm the existence of a dorsoventral asymmetry in the distribution of Tube at the embryo surface. Tube is more highly concentrated along the ventral surface than the dorsal surface; the difference in concentration is two-fold, as assayed by quantitation of staining intensity. Higher magnification ventral views reveal that Tube clusters along the embryo surface. These clusters or aggregates are approximately 2 mm in diameter and are located between adjacent ventral nuclei at sites of membrane invagination. On the dorsal side of the embryo, clusters of Tube are less readily detectable (Towb, 1998).

To determine whether the asymmetric distribution of Tube depends on Toll-mediated signaling, Tube localization was examined in genetic backgrounds that either inactivate or constitutively activate the signaling pathway. The gd2 mutation blocks Toll activation, whereas the Toll10b mutation alters the Toll extracellular domain so as to constitutively activate the receptor throughout the embryo. Both mutations eliminate the dorsoventral asymmetry in Tube staining observed in the wild type. However, Tube is more highly concentrated at the periphery of Toll10b embryos than gd2 embryos. The Toll10b and gd2 results support the model that activated Toll directs recruitment of Tube to the embryo surface. The asymmetry in Tube localization in the wild type is not, however, as striking as that observed for Cactus or Dorsal. Therefore, the distribution of Tube was also examined in P[Toll10b-bcd] embryos, in which Toll-mediated signaling is ectopically oriented along the anteroposterior axis. Females carrying the P[Toll10b-bcd] transgene express high levels of the Toll10b cDNA fused to the bicoid (bcd) 3'UTR and consequently produce embryos in which the bcd sequences localize the Toll10b mRNA to the anterior pole. Tube concentrates at the plasma membrane in and near the anterior pole of P[Toll10b-bcd] embryos. Moreover, clusters of Tube are prominent anteriorly on both the dorsal and ventral surfaces of such embryos. The Toll10b protein expressed from the P[Toll10b-bcd] construct also forms cell surface aggregates at the anterior pole. Comparison of the patterns of Tube and Toll localization in P[Toll10b-bcd] embryos indicates that the Toll aggregates colocalize with the Tube aggregates (Towb, 1998).

Since Tube and Pelle are thought to interact in embryos, it was reasoned that Pelle, like Tube, might localize to sites of Toll activation. Using a polyclonal anti-Pelle serum to stain wild-type embryos, it was found that Pelle is distributed throughout the embryo. A fraction of Pelle localizes to the surface of embryos, but there is no significant asymmetry in this distribution of Pelle across the dorsoventral axis. If Pelle undergoes a signal-dependent membrane association in wild-type embryos, this association is too short-lived or involves too few of the Pelle molecules in the embryo to be readily detectable. However, since localization studies had indicated that signal-dependent protein relocalization is enhanced in embryos expressing high levels of Toll10b protein at the anterior pole, Pelle localization was also examined in embryos generated by P[Toll10b-bcd] females. A large fraction of the Pelle protein localizes to the anterior end of P[Toll10b-bcd] embryos, indicating a significant recruitment or stabilization of Pelle protein at this pole. Confocal cross sections have revealed that the anterior-localized Pelle is predominantly at the embryonic periphery, possibly in association with the plasma membrane. High levels of activated Toll thus promote localization of both Tube and Pelle to the surface of the embryo. Since Tube localization at the anterior end of P[Toll10b-bcd] embryos mirrors that in the ventral portion of wild-type embryos, it is proposed that the localization of Pelle in P[Toll10b-bcd] embryos similarly reflects a relocalization occurring in wild-type embryos in response to Toll activation (Towb, 1998).

Biochemical interactions are described between recombinant Toll, Pelle and Tube that provide insights into early events in activation of the signaling cascade. A tertiary complex exists prior to activation. The Pelle-Toll complex is required to suppress that kinase activity of Pelle. Pelle binds directly to a region within the Toll intracytoplasmic domain, providing the first evidence that these two evolutionarily conserved molecules physically interact. Pelle contains an N-terminal putative regulatory domain, consisting largely of a region with significant similarity to the consensus death domain, and a C-terminal catalytic domain. To determine whether either (or both) of these regions are required for the interaction with the intracellular domain of Toll (Toll IC), the Pelle N terminus (Pelle-R) and C terminus (Pelle-C) were produced separately by in vitro translation, and then tests were carried out to see if these molecules could bind any of several GST-Toll IC derivatives. The results indicate that the Pelle N terminus is incapable of binding any of the GST-Toll derivatives, whereas the catalytic region binds efficiently to both GST-Toll and GST-Toll ICNae (a derivative lacking the C-terminal ID but containing the entire IL-1R homology region). The Toll C-terminal inhibitory domain (ID) is neither necessary nor sufficient for this interaction (Shen, 1998).

It is thought that upon activation Pelle is autophosphorylated, and that this prevents binding to Toll as well as Tube. Autophosphorylation occurs in the N-terminal, death-domain-containing region of Pelle, a region dispensable for binding to Toll but required for enzymatic activity. Pelle phosphorylates Toll, within the region required for Pelle interaction, but this phosphorylation can be blocked by a previously characterized inhibitory domain at the Toll C terminus. These and other results allow for the proposal of a model by which multiple phosphorylation-regulated interactions between these three proteins lead to activation of the Dorsal signaling pathway (Shen, 1998).

It is proposed that the intracytoplasmic IL-1R homology domain of the Toll receptor initially interacts directly with unphosphorylated Pelle, and that the Toll ID helps down regulate kinase activity. Tube is also recruited to the complex through its interaction with Pelle and/or Toll, forming the Toll/Pelle/Tube ternary complex (Pelle inactive). Binding of Spätzle to Toll induces Toll dimerization, and the Toll cytoplasmic domain is modified through conformational changes or proteolysis of the Toll inhibitory domain, allowing activation of kinase activity (Pelle active). Active Pelle then phosphorylates multiple substrates, including itself, Toll and Tube. This causes disruption of the Toll/Pelle/Tube complex, freeing Pelle to phosphorylate unknown downstream targets, eventually resulting in Cactus phosphorylation/degradation and Dorsal phosphorylation (e. g., by protein kinase A [PKA] and possibly Pelle itself] and nuclear translocation. Phosphorylated Tube may also translocate with Dorsal and function as a transcriptional coactivator (Shen, 1998).

The direct interactions described between unphosphorylated Pelle, Toll and Tube are consistent with the existence of a ternary complex at the plasma membrane. Pelle interacts with Toll via residues in its catalytic domain, and with Tube via Pelle's N-terminal death domain: both interactions can occur simultaneously. An important question is whether the ternary complex forms independent of signaling. Previous studies have shown that the artificial recruitment of Pelle or Tube to the plasma membrane can initiate the signaling pathway independent of ligand binding. But it is not clear whether it is recruitment to the membrane per se that results in activation, or the dimerization of the Torso fusion proteins employed in these previous studies. A possible mechanism for Pelle activation is simply dimerization, induced naturally, it is suggested, by conformational changes in the ternary complex that occur following ligand binding. How might such changes be induced? There is considerable indirect evidence suggesting that Toll molecules interact: an attractive model posits that ligand binding induces dimerization or even aggregation. It is suggested that this leads to activation of signaling, i.e., of Pelle activity, by either (or both) of two related mechanisms: (1) oligomerization of Toll receptors increases the local ternary complex concentration and hence Pelle concentration, thereby favoring Pelle dimerization and activation by simple mass action; (2) ligand-induced Toll self-association causes a conformational change in the intracytoplasmic domain such that the ID is displaced, thereby facilitating Pelle activation, again perhaps by dimerization. A speculative possibility is that the ID is actually cleaved upon activation. The product of the strong dominant gain-of-function allele Toll 10b, which contains a single C to Y change in its extracellular domain, has been found in a partially proteolyzed form, such that full-length Toll 10b is associated with a truncated form lacking most or all of its extracellular domain as well as likely sequences from the very C terminus, i.e., the ID. Perhaps relevant to this, a putative PEST degradation sequence is situated between the IL-1R homology region and the ID. It is intriguing that the structure of this truncated product is similar to mammalian IL-1RAcp, which functions in IRAK activation during IL-1 signaling. In any event, it is proposed that IL-1R homology domain interactions activate Pelle via the direct, phosphorylation-sensitive protein-protein interactions described in this paper (Shen, 1998).

In the Drosophila embryo the nuclear localization of Dorsal, a member of the Rel family, is regulated by an extracellular signal, which is transmitted to the interior of the egg cell by a cascade of proteins involving the novel protein Tube and the protein kinase Pelle. The activation mechanism of Tube and Pelle and the interaction between these two components have been analyzed. Both proteins, although having different biochemical activities, are activated by the same mechanism. Membrane association alone (driven by creating Torso-Pelle or Torso-Tube fusion proteins) is not sufficient, but oligomerization (driven by creating constitutively active Torso in these fusion proteins) is required for full activation of Tube and Pelle. By deletion analysis the domains of Tube and Pelle mediating the physical interaction and the signaling to downstream components has been determined. In order to investigate the link between Pelle and the target of the signaling cascade (the Dorsal/Cactus complex) a novel but evolutionary conserved protein termed Pellino was isolated and characterised. Pellino associates with the kinase domain of Pelle (Grosshans, 1999).

Pellino is a protein with 424 amino acids. It is highly conserved to a protein of unknown function in C. elegans, with about 50% identity and 70% homology. Mouse and human homologs have been identified. Pellino is expressed during all stages of develoment, coded for by a transcript of 3kb. In addtion, there is a oocytic transcript of 1.9kb found only in females and in eggs. Pellino does not bind to a catalytically inactive form of the Pelle kinase and Pellino is not phosphorylated by Pelle. These observations indicate that Pellino, unlike Tube, is not a phosphorylation substrate of Pelle, but that it probably only binds to the autophosphorylated form of Pelle. Tube and Pellino are shown ot bind to different domains of Pelle. Pellino is probably involved in the signaling steps downstream of Pelle. Pellino could stabilize the activated state of Pelle after the dissociation from Tube oligomers or could mediate the interaction to the downstream phosphorylation substrate of Pelle. Alternatively, the signal transduction by activated Pelle oligomers may occur through the binding of Pellino and other downstream components without any phosphorylation of them by Pelle. Future studies of Pellion and the identification of mutations in the pellino gene will allow a better understanding of the molecular nature of the link between Pelle and the Dorsal/Cactus complex (Grosshans, 1999).

Dorsoventral polarity in the Drosophila embryo is established through a signal transduction cascade triggered in ventral and ventrolateral regions. Activation of a transmembrane receptor, Toll, leads to localized recruitment of the adaptor protein Tube and protein kinase Pelle. Signaling through these components directs degradation of the IkappaB-like inhibitor Cactus and nuclear translocation of the Rel protein Dorsal. Pelle mediates feedback regulation in the Drosophila Toll signaling pathway. Pelle functions to downregulate the signal-dependent relocalization of Tube. Inactivation of the Pelle kinase domain, or elimination of the Tube-Pelle interaction, dramatically increases Tube recruitment to the ventral plasma membrane in regions of active signaling. A large collection of pelle alleles has been characterized in this study, leading to an identification of the molecular lesions in these alleles and their effects on Pelle autophosphorylation, Tube phosphorylation and Tube relocalization. These results point to a mechanism operating to modulate the domain or duration of signaling downstream from Tube and Pelle (Towb, 2001).

An activity of Pelle, possibly direct phosphorylation of Tube, is required to disaggregate membrane-associated Tube clusters. In pelle mutant embryos, Tube clustering along the ventral midline is greatly enhanced over that seen in wild-type embryos. Elimination of either of two wild-type activities of Pelle -- Tube binding or protein kinase function -- is sufficient for this enhancement (Towb, 2001).

Pelle can phosphorylate Tube in vitro. Furthermore, the pll⁰⁷⁴ mutation, which blocks Tube phosphorylation but not Pelle autophosphoryation, enhances Tube clustering. It is suggested, therefore, that in the wild-type embryo Pelle-mediated phosphorylation of Tube causes dissociation of multiprotein complexes containing Tube. The weak gradient of Tube seen in the wild type would thus represent a balance between Tube recruitment to clusters and Tube release from clusters upon Pelle-catalyzed phosphorylation (Towb, 2001).

Although the idea that Pelle directly phosphorylates Tube to disrupt Tube-containing complexes is favored, alternative mechanisms for regulating complex stability are possible. For example, Pelle might phosphorylate and activate an unidentified downstream target that would then disaggregate the Tube-containing complex. Distinguishing between these possibilities is made difficult by the fact that all known mutations that prevent the association of Tube and Pelle also block Pelle activation and hence downstream function. For instance, the tub² mutation, which prevents interaction with Pelle, completely blocks signaling to Dorsal. Similarly, mutations in Pelle that do not affect the catalytic domain, but that alter the Tube binding domain, disrupt Pelle function (Towb, 2001).

Nearly all the Pelle missense mutations sequenced in this study map to the kinase domain rather than the death domain, the site of interaction with Tube. A possible explanation for this bias became apparent in the structural analysis of the interaction between Tube and Pelle. A combination of crystallographic studies and mutational analyses has revealed that the Tube death domain can productively interact with two different sets of surface residues in the Pelle death domain. One would therefore predict that most Tube binding sites in the Pelle death domain would be redundant in function and that such sites would not mutate to a loss-of-function phenotype. Thus, mutations that block signaling would most frequently map to the death domain of Tube, but outside the death domain in Pelle, as is in fact the case (Towb, 2001).

Extensive genetic characterization of pelle mutations led to the classification of several alleles as recessive antimorphs. The term antimorphic indicates that the presence of this allele is more detrimental to development than a complete absence of the functional gene. Most of the pelle alleles classified as strong antimorphs alter residues in or near a part of the kinase structure known as the activation loop. Such activation loops often contain residues that are targets for upstream, activating kinases. In the case of the protein kinase ERK2, for example, phosphorylation of residues in the activation loop alters the conformation of the active site cleft, affecting ERK2's ability to bind ATP and changing the conformation of the substrate-recognition region. By analogy, the antimorphic effect of several pelle mutations may be due to their binding and sequestering either an upstream activating kinase for Pelle or a Pelle substrate (Towb, 2001).

Regulation of protein-protein interaction through phosphorylation has been widely documented in signal transduction pathways. In visual signal transduction, for example, modulation of the phosphorylation state of phosducin regulates the interaction of phosducin and the ßgamma subunits of transducin. In the case of Tube and Pelle, such a change in phosphorylation state could be catalyzed by Pelle itself. This could explain why a catalytically inactive form of Pelle interacts more strongly with Tube in the yeast two-hybrid system than does the wild-type Pelle (Towb, 2001).

Phosphorylation of Tube by Pelle could act negatively on the pathway, breaking apart Tube-containing complexes, as well as weakening the Tube/Pelle interaction. Since Pelle acts downstream of Tube and requires Tube-mediated targeting to the membrane for activation, both of these feedback mechanisms could act to turn Pelle activity down and decrease downstream signaling. This regulation may serve to increase the fidelity of downstream signal transduction. High, medium, and low nuclear levels of the Dorsal transcription factor control the transcription of different sets of genes in highly circumscribed ventral-to-dorsal territories in the early embryo. Feedback mechanisms in the signal transduction pathway downstream of Toll may have evolved to ensure that proper nuclear Dorsal levels are maintained in the correct spatial regions of the embryo (Towb, 2001).

In considering negative regulation of Toll signaling, it is necessary to take into account that signal transduction is taking place during the highly rapid nuclear division cycles of early Drosophila embryogenesis. The Dorsal nuclear localization gradient does not persist during mitosis, but rather reforms after every nuclear division. Furthermore, Tube becomes associated with nuclei during each mitosis. Negative feedback may therefore be required to return the signaling cascade downstream of Toll to a starting state, setting the stage for reformation of the Dorsal gradient in the next interphase. In this regard, it is noted that the activation of both Spätzle and Easter, which act upstream of Toll, appears to be subject to a distinct form of negative feedback regulation (Towb, 2001).

Although the phenomenon of Tube gradient enhancement could, as outlined above, reflect a negative feedback mechanism, it is also possible that Tube release from clusters and Tube/Pelle complex dissociation are mechanisms that act to promote or spatially regulate downstream signaling. Activated Tube molecules might need to diffuse away from a signaling complex, or other components might need to cycle through steps of activation and deactivation, in order for downstream signaling to occur. Alternatively, if the Toll receptor is only activated in a narrow ventral stripe, the diffusion of activated Tube and Pelle could serve to establish a gradient of signaling away from the ventral midline, and thus fine tune the gradient of Dorsal nuclear translocation (Towb, 2001).

The mechanism proposed for downregulation of the dorsoventral pathway signal may function in mammals. The adaptor protein, MyD88, has been reported to bind more tightly to IRAK when the IRAK kinase domain has been inactivated, similar to the situation with Tube and Pelle. Therefore, upon activation, IRAK may phosphorylate MyD88 and cause the signaling complex to dissociate. This mechanism of regulating death domain interactions through phosphorylation, would thus be another aspect of the host defense system inherited from the common ancestor of flies and humans (Towb, 2001).

MyD88 is an adaptor protein that interacts physically with Toll and with the kinase Pelle and functions upstream of Tube and Pelle

Drosophila MyD88 is an adapter in the Toll signaling pathway that associates with both the Toll receptor and the downstream kinase Pelle. Expression of MyD88 in S2 cells strongly induces activity of a Drosomycin reporter gene, whereas a dominant-negative version of MyD88 potently inhibits Toll-mediated signaling. MyD88 associates with the death domain-containing adapter Drosophila Fas-associated death domain-containing protein (FADD), which in turn interacts with the apical caspase Dredd. This pathway links a cell surface receptor to an apical caspase in invertebrate cells and therefore suggests that the Toll-mediated pathway of caspase activation may be the evolutionary ancestor of the death receptor-mediated pathway for apoptosis induction in mammals (Horng, 2001).

A BLAST search of the Drosophila genome identified the sequence encoding MyD88, a Drosophila homolog of human MyD88. Similar to its human homolog, Drosophila MyD88 contains an N-terminal death domain, an intermediate domain, and a TIR domain. However, unlike human MyD88, Drosophila MyD88 contains an additional 81 amino acids preceding the death domain and a 162-aa-long C-terminal region following the TIR domain (Horng, 2001).

Transfection of MyD88 into Drosophila S2 cells potently induces a Drosomycin reporter gene but not an Attacin reporter gene. This preferential ability to induce an antifungal gene is similar to that of Toll 10b, a constitutively active form of Toll, and suggests that MyD88 may be a component of the Toll-Tube-Pelle-Cactus-Dif signaling pathway. Previous studies have demonstrated that Toll-mediated Drosomycin induction requires the nuclear translocation of Dif. Dif is normally retained in the cytoplasm by the IkappaB inhibitor Cactus and is released only in response to signal-dependent degradation of Cactus. To test whether MyD88-mediated Drosomycin induction also depends on Cactus degradation, a Cactus mutant was constructed that contains mutations of the conserved serine residues that, in mammalian IkappaB, are the targets of signal-dependent phosphorylation. A Cactus mutant inhibits Drosomycin induction by MyD88 and, as expected, by Toll. This result indicates that, similar to Toll, MyD88 regulates Drosomycin induction through the Cactus-dependent pathway (Horng, 2001).

For further analyses, various deletion mutants of MyD88 were generated. Two of the deletion mutants, one containing the TIR domain and the C-terminal domain (amino acids 237-537) and another containing the intermediate, TIR, and C-terminal domains (amino acids 176-537), activate the Drosomycin reporter weakly (10-fold) in comparison to full length MyD88, indicating that the intact protein is required for optimal activity. However, the fact that these truncation mutants can still induce signaling is surprising, since they lack the death domain that mediates interactions with downstream signaling components. Moreover, similar analyses of human MyD88 have shown that a combination of the death domain and the intermediate domain is sufficient to induce signaling activity comparable to that of the wild-type protein. An equivalent truncation of dMyD88 (amino acids 1-237) retains no residual activity despite being well expressed, suggesting that there are some differences in domain function between human and Drosophila MyD88 proteins (Horng, 2001).

To determine whether MyD88 is a component of the Toll signaling pathway, attempts were made to identify a deletion mutant that would have dominant-negative activity. Therefore, three MyD88 deletion mutants that do not activate the Drosomycin reporter were tested for their ability to inhibit Toll-mediated Drosomycin induction. The strongest inhibitor was the death domain- and middle domain-containing construct (amino acids 1-237), which at low concentrations potently inhibits Toll-mediated Drosomycin induction in a dose-dependent manner (Horng, 2001).

To order MyD88 in the pathway with respect to Pelle, MyD88 was tested for its ability to be inhibited by PelleN, a dominant-negative form of Pelle that consists of the N-terminal death domain-containing region of Pelle. MyD88, like Toll, is strongly inhibited by PelleN. MyD88, however, does not inhibit Pelle, demonstrating that, similar to the mammalian pathway, MyD88 functions upstream of Pelle (Horng, 2001).

To further establish MyD88 as a component of the Toll pathway, whether MyD88 interacts with Toll was tested by coimmunoprecipitation assays. The TIR domain-containing MyD88 construct is detected in anti-Toll immunoprecipitates. Interestingly, when cotransfected with Toll 10b, MyD88 reproducibly appears as two distinct bands -- a slower migrating upper band that may correspond to phosphorylated MyD88 construct and a faster migrating lower band. The predominant form of MyD88 detected in immunoprecipitates is the faster migrating species. MyD88 therefore associates with Toll, presumably through TIR domains, and is a component of the active receptor complex (Horng, 2001).

Because human MyD88 associates with IRAK through death domains, a likely immediate downstream target of MyD88 is the IRAK homolog Pelle. Interaction between the death domain-containing dMyD88 construct (amino acids 1-237) and Pelle was examined. MyD88 is detected in Pelle immunoprecipitates, indicating that MyD88 interacts with Pelle, presumably through their death domains (Horng, 2001).

These results therefore demonstrate that MyD88 is an adaptor in the Toll signaling pathway downstream of the receptor and upstream of Pelle. From genetic analyses, the adaptor protein Tube has also been implicated to be downstream of Toll and upstream of Pelle in the Toll signaling pathway. The death domain of Tube also interacts with Pelle. Because Tube and MyD88 also contain death domains that could potentially mediate their interaction, tests were performed for association between these two proteins in immunoprecipitation assays; Tube and MyD88 do indeed interact. Therefore, MyD88 and Tube both function as adaptors downstream of Toll, exist in the same active complex along with Pelle, and are probably both involved in the recruitment and/or activation of Pelle. Understanding functional differences between these two adapters will require further analysis (Horng, 2001).

To identify other potential downstream targets of MyD88, a search of the Drosophila genome was performed for other sequences that encode death domain-containing proteins that may interact with MyD88. One such sequence encodes a protein with a death domain as well as a death effector domain and appears to be a homolog of mammalian FADD. This cDNA has been identified and named FADD (Hu, 2000). Whether FADD can interact with MyD88 was tested. Lysates from S2 cells transfected with MyD88 were incubated with anti-Flag beads to immunoprecipitate FADD, and immunoprecipitates were blotted with anti-V5 antibody to look for associated MyD88. A strong band corresponding to MyD88 was observed, indicating that MyD88 can interact with FADD through death domains. Overexpression of FADD in S2 cells, however, does not lead to activation of either the Drosomycin or Attacin reporters (Horng, 2001).

Mammalian FADD is recruited to the tumor necrosis factor receptor complex through homophilic death domain interactions with the adapter TNFR-associated death domain-containing protein (TRADD). In turn, FADD recruits procaspase-8 through homophilic death effector domain associations. It is speculated that Drosophila FADD may likewise recruit a Drosophila caspase to the Toll receptor complex. A potential candidate caspase is Dredd, an apical caspase with a long prodomain shown to be essential for induction of antibacterial genes. Indeed, analysis of immunoprecipitated lysates from cells cotransfected with Drosophila FADD, and either full length Dredd or the death effector domain of Dredd showed strong association of Dredd with FADD. A second study (Hu, 2000) has also shown interaction of dFADD with Dredd (Horng, 2001).

Thus Drosophila MyD88 is an adapter in the Toll signaling pathway. MyD88 associates with both Toll and Pelle and functions upstream of Pelle. Tube is known from genetic studies to be an adapter in the Toll pathway that functions upstream of Pelle. Why Toll should signal through MyD88 and Tube, two receptor-proximal adapters with seemingly similar functions, is not yet clear. MyD88 associates with the receptor Toll as well as the downstream adapter FADD, which in turn interacts with the apical caspase Dredd. Because caspases are essential executioners of the apoptotic machinery in organisms from nematodes to mammals, and because Dredd has been shown to be involved in apoptosis during Drosophila development, it is possible that Toll-1 or some of the other eight Tolls that exist in Drosophila may induce apoptosis (or another Dredd-dependent pathway) through the MyD88/dFADD/Dredd pathway in a cell-type specific and/or developmental stage-specific manner. The pathway comprised of Toll, MyD88, dFADD, and Dredd would be the first description of a pathway in invertebrates that links a cell surface receptor to an apical caspase. Such a pathway, if it exists, would enable extracellular stimuli, perhaps ligands secreted by other cells during development or pathogen-derived products during infection, to instruct invertebrate cells to undergo cell death. In addition, the Toll/MyD88/dFADD/Dredd pathway is remarkably similar to that activated by the receptors of the tumor necrosis factor receptor (TNFR) superfamily in mammals, in which FADD-mediated recruitment of caspase-8 leads to induction of apoptosis. Since the Drosophila genome does not encode any cell surface receptors homologous to TNFRs, it appears that the Toll/MyD88/dFADD/Dredd pathway is the evolutionary ancestor of the mammalian death receptor pathways. This possibility is further supported by the recent finding that human TLR2 can induce apoptosis through the MyD88/FADD/Caspase-8 pathway (Horng, 2001).

A heterotrimeric death domain complex involving Tube, Pelle and Myd88

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).

Myd88 interacts with Pelle and Tube in the establishment of dorsoventral pattern in the Drosophila embryo

In Drosophila, the dorsoventral axis is set up by the action of the dorsal group of genes and cactus, which have been ordered genetically in a linear pathway. krapfen (kra) has been identified and characterized as a new member of the dorsal-group genes. kra (accepted FlyBase name: Myd88) encodes for the Drosophila homolog of MyD88, an adapter protein operating in the mammalian IL-1 pathway. Epistasis experiments reveal that kra acts between the receptor Toll and the cytoplasmic factor Tube. There is a direct interaction between Kra 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, Kra associates with Pelle and Tube, in an heterotrimeric complex (Charatsi, 2003).

Whether Kra participates in the activation of the Toll receptor or whether Kra cooperates in the signal transduction downstream of Toll was investigated. The effect of loss of maternal kra was tested in a dominant Toll background, Toll^9Q, which is a ligand-independent gain-of-function allele of Toll. Embryos laid by Toll^9Q heterozygous flies show a strongly ventraliz ed phenotype due to the constitutively active Toll that signals throughout the embryo circumference. Embryos laid by females with the genotype kra¹/kra¹; Toll^9Q/TM3 show a complete dorsalized phenotype. Thus, kra suppresses the constitutive Toll signal, indicating that Kra acts downstream of the Toll receptor. This finding suggests that Kra operates in the cytoplasmic compartment of the Drosophila early embryo. In order to place Kra upstream or downstream of the cytoplasmic protein Tube, the phenotype of kra embryos was analyzed after microinjection of the gain-of-function construct of Tube, pBtor⁴⁰²¹Tube. In this construct, the intracellular kinase domain of a gain-of-function allele of the receptor tyrosine kinase torso and is replaced by the tube coding sequence. pBtor⁴⁰²¹ fusions have been used previously to show that Tube operates upstream of Pelle. Whereas uninjected kra embryos develop only dorsal epidermis, kra embryos injected with pBtor⁴⁰²¹Tube RNA can specify ventrolateral fates and restore ventrolateral pattern elements, such as ventral denticle belts and Filzkörper, that are never observed in kra mutant embryos. Through these methods kra can be placed downstream of Toll and upstream of tube (Charatsi, 2003).

The genetic positioning of kra between Toll and tube, however, does is not informative as to the physical interactions that take place during signal transduction. In order to investigate the molecular role of kra, a yeast two hybrid assay was performed. kra 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 tumour 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. In the yeast two hybrid assay, wild type Kra as well as Kra⁵⁶, which is an EMS allele carrying a missense mutation within the TIR domain, were both able to interact strongly with Tube. This indicates that the TIR domain is not required for a Kra-Tube interaction (Charatsi, 2003).

Pelle interacts with Tube through death domain association. In the same yeast two hybrid experiment no direct interaction was found between Pelle and Kra. This suggests that Tube could mediate the formation of a complex by association with both Pelle and Kra (Charatsi, 2003).

The epistasis experiment placed Kra downstream of the receptor Toll. Both Toll and Kra contain TIR domains, which could potentially mediate their interaction. Additionally, the kra⁵⁶ allele which shows a dorsalized phenotype presumably caused by a defective TIR domain, strongly suggests that this cytoplasmic domain plays an essential role in signal transduction. An interaction between Kra and Toll, which is not necessarily direct, is supported by immunoprecipitation experiments in which Kra/dMyD88 coimmunoprecipitates with Toll. However, in the yeast two hybrid assay, no direct interaction was found between the TIR cytoplasmic domain of Toll and Kra (Charatsi, 2003).

In order to understand how the signal is transduced to Kra through Toll, the possibility that Kra homodimerises was investigated. In mice, MyD88 was shown to form homodimers in vivo through death domain-death domain and TIR-TIR interactions. Kra does not homodimerize in yeast two hybrid assay nor in immunoprecipitation experiments (Charatsi, 2003).

Therefore Drosophila Myd88 plays a crucial role in the dorsoventral patterning of the embryo through the Toll pathway. Embryos mutant for three new kra alleles display a dorsalized phenotype. Two of these alleles, kra¹ and kra², carry large deletions removing the ATG, and represent null alleles. Embryos from homozygous females carrying these alleles are completely dorsalized, whereas the EMS missense allele, kra⁵⁶, causes a partial dorsalization. Since the EP insertions do not cause the dorsalization of the embryo, it is evident that the Toll pathway is more sensitive to the amount of dMyd88 in the immune response than in the dorsoventral system (Charatsi, 2003).

RNA injection as well as two hybrid data reveal that Myd88/Kra associates with and activates Tube, but not Pelle. Thus, Tube interacts both with Kra and Pelle. A model is proposed in which Tube as a central molecule binds to Kra and Pelle to form a heterotrimeric complex which is essential for the signal transduction to downstream components. This view is supported by immunoprecipitation experiments in which Kra/MyD88 was shown to coimmunoprecipitate with Pelle. These associations are presumably mediated by the death domains present in all these proteins. This model is supported by studies on other death domain containing proteins in the apoptotic signalling pathways that form multimeric complexes (Charatsi, 2003).

Immunoprecipitation experiments with the Drosophila and the murine Myd88 suggest that Kra associates with the receptor Toll to transduce the extracellular signal. However, Kra does not interact with Toll in the two hybrid assay. Moreover, in contrast to the murine Myd88, Kra does not homodimerize. These findings together with the comparison of the sequences point out that despite the homology shared by MyD88 and Kra, functional differences may exist. Unlike MyD88, Kra contains an additional 81 amino acids preceding the death domain and a 162 amino-acid long C-terminal region, both regions are as yet of unknown function. It is therefore concluded that homodimerization plays no role in the mechanism of signal transduction (Charatsi, 2003).

The negative results of the two hybrid experiments could be explained by the interaction of Kra and Toll mediated by an unknown component. Since no apparent signalling domains can be identified either in Kra or in Toll, it is likely that additional proteins are recruited. Alternatively, Kra could interact only with an activated form of Toll. It has been suggested that transduction of the signal from Toll to downstream components could be dependent on a conformational change of the Toll receptor upon ligand binding, an idea that is supported by structural studies of the Toll receptor. However, this alternative can not be tested by a yeast two hybrid assay (Charatsi, 2003).

The Toll pathway shows striking similarities to the IL-1 pathway. Toll in Drosophila and the IL-1R in mammals are related transmembrane receptors. As is the case during Toll signalling, binding of IL-1 to the receptor complex IL-1R/IL-1AcP (Interleukin-1 Receptor/Interleukin-1 Accesory Protein) leads to activation of transcription factors of the Rel family. Upon ligand binding MyD88 associates with IL-1AcP and mediates the formation of the receptor complex. In the Drosophila Toll pathway and the homologous mammalian pathway most components are conserved. However, since Tube has been identified within only the Drosophila genus, it had previously been proposed that Tube is the functional homolog of the vertebrate MyD88, operating between Toll and Pelle. The identification of a functional Myd88 in Drosophila shows that Tube is an additional factor. Further studies on the interaction between Tube and Kra/dMyd88 should help to understand the enigmatic role of Tube in the establishment of the dorsal gradient in the early Drosophila embryo (Charatsi, 2003).

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