BIOLOGICAL OVERVIEW

The proper alignment or polarity of the fly along its dorsal-ventral axis is the result of cooperative functioning among a number of genes collectively termed the Dorsal group. tube is one component of this group. Dorsal group proteins are supplied maternally; that is, they are present in the egg prior to fertilization. Their functioning leads to the activation of Dorsal protein, a transcription factor responsible for structuring dorsoventral polarity. The cascade of events begins with signals from the Toll receptor. These are transmitted via Tube and Pelle to Dorsal. Dorsal activation must also be accompanied by the destruction of Cactus, which otherwise would bind Dorsal, in effect imprisoning it in the cytoplasm and thereby rendering it inactive. Once freed (activated), Dorsal migrates from cytoplasm to nucleus where it then functions to activate and repress genes involved in dorsoventral polarity, including decapentaplegic, twist and snail.

Tube accomplishes two critical tasks. The first involves its interaction with Pelle, leading to the destruction of Cactus. Signal-dependent degradation of Cactus does not require the presence of Dorsal, indicating that Cactus degradation is a direct response to Pelle signaling. But Cactus degradation requires both Tube and Pelle (Belvin, 1995). So what gets Tube into the act?

Tube is membrane associated in the absence of Toll. Upon activation of Toll, Tube activates Pelle, a serine-threonine kinase. The only known phosphorylation target of Pelle is Tube (Grosshans, 1994). Each of these interactions leads to the destruction of Cactus, which in the case of I kappa B, the vertebrate homolog of Cactus, is ultimately carried out by ubiquitin, the cell's machinery for protein degradation.

The second function of Tube involves an interaction with Dorsal. Tube accompanies Dorsal on its migration into the nucleus (having acted with Pelle to destroy Cactus's cytoplasmic hold on Dorsal). Expression of tube does not by itself affect the localization of Dorsal. Toll's part is essential. Activation of Toll enhances nuclear localization of DL and increases dl transcriptional activation. When tube is coexpressed with Toll, dl transcriptional activity is further enhanced. In addition, coexpression of tube and Toll resulting in the nuclear localization of Dorsal, results in the nuclear localization of Tube as well.

Quite possibly Tube is a chaperone for Dorsal, accompanying it into the nucleus. In addition, the effect of Tube on dorsal transcription suggests that Tube might be a transcription factor acting to regulate dorsal transcription (Norris, 1995). Thus Tube has a cytoplasmic role in the activation of Pelle and the destruction of Cactus, and a subsequent role in association with Dorsal in Dorsal's migration to the nucleus, where it might serve to activate dorsal transcription.

Weckle is a zinc finger adaptor of the toll pathway in dorsoventral patterning of the Drosophila embryo

The Drosophila Toll pathway takes part in both establishment of the embryonic dorsoventral axis and induction of the innate immune response in adults. Upon activation by the cytokine Spätzle, Toll interacts with the adaptor proteins DmMyD88 and Tube and the kinase Pelle and triggers degradation of the inhibitor Cactus, thus allowing the nuclear translocation of the transcription factor Dorsal/Dif. weckle (wek) has been identified as a new dorsal group gene that encodes a putative zinc finger transcription factor. However, its role in the Toll pathway was unknown. This study isolated new wek alleles and demonstrated that cactus is epistatic to wek, which in turn is epistatic to Toll. Consistent with this, Wek localizes to the plasma membrane of embryos, independently of Toll signaling. Wek homodimerizes and associates with Toll. Moreover, Wek binds to and localizes DmMyD88 to the plasma membrane. Thus, Wek acts as an adaptor to assemble/stabilize a Toll/Wek/DmMyD88/Tube complex. Remarkably, unlike the DmMyD88/tube/pelle/cactus gene cassette of the Toll pathway, wek plays a minimal role, if any, in the immune defense against Gram-positive bacteria and fungi. It is concluded that Wek is an adaptor to link Toll and DmMyD88 and is required for efficient recruitment of DmMyD88 to Toll. Unexpectedly, wek is dispensable for innate immune response, thus revealing differences in the Toll-mediated activation of Dorsal in the embryo and Dif in the fat body of adult flies (Chen, 2006).

Through a BLAST search, three zinc finger-containing genes, CG17568, CG10366, and CG6254, were identified as wek paralogs in the Drosophila genome. In addition to having high homology in the C-terminal zinc finger motifs, Wek also shows homology of 58%-65% and identity of 30%-43% in the N terminus with these three genes. This region is referred to as the WekN domain (amino acids 1-103) and the C-terminal region as the WekC domain (amino acids 273-470) that contains the six zinc fingers. The rest of Wek was designated as WekM domain (amino acids 104-272) . Although WekC shows high homology with several zinc finger proteins in mammals, no clear wek ortholog was identified in mammals using WekN and WekM (Chen, 2006).

Interaction between the TIR domains of Toll and DmMyD88 has been suggested by coimmunoprecipitation experiments in cultured cells. However, no interaction has been detected between DmMyD88 and the Toll intracytoplasmic domain in yeast two-hybrid assays, although interaction is detected between DmMyD88 and Tube. In addition, overexpression of the TIR domain of DmMyD88 leads to strong activation of the pathway, instead of behaving like a dominant negative, as one would expect for a domain-mediating interaction with upstream components of the pathway. Thus, there are indications that the situation may be more complex than initially assumed and involve a supplementary factor in the receptor complex. This study describes a zinc finger protein that functions as an adaptor in the Toll pathway. The data clearly establish that efficient recruitment of DmMyD88 to Toll in the embryo requires Wek and that Wek is part of the Toll receptor complex. This model is supported by coimmunoprecipitations in Drosophila cultured cells, immunolocalization in embryos, and finally genetics, as embryos laid by wek mutant females exhibit similar phenotypes as other Toll pathway mutants. Furthermore, strong genetic interactions are detected between wek and other genes of the Toll pathway. Interestingly, it was noticed that Wek also interacts with Toll-9 and to a lesser extent Toll-5, but not with other members of the Toll family. Toll, Toll-9, and Toll-5 are the only members of the family that are able to activate the Toll pathway in tissue culture cells, and hence there is a perfect correlation between the capacity to interact with Wek and the activation of the pathway (Chen, 2006).

Because DmMyD88 associates with active Toll, the observation that Wek is required to localize DmMyD88 to the cell surface even when Toll is active indicates that the physical interaction between active Toll and DmMyD88 alone might not be stable. This together with a series of binding results leads to a proposal that, before Toll activation, Wek bridges Toll and the DmMyD88/Tube complex to assemble into a large Toll/Wek/DmMyD88/Tube presignaling complex on the membrane. Upon Toll activation, Toll can now simultaneously associate with both adaptors (DmMyD88 and Wek) to assemble a much more stable Toll/Wek/DmMyD88/Tube complex that leads to the differential association and activation of Pelle. This model not only explains why DmMyD88 is still membrane associated when Toll is inactive on the dorsal side of the embryo but also explains why Tube and DmMyD88 distribute asymmetrically along the dorsoventral axis with increased concentration along the ventral surface. In the absence of Wek, the weak association between active Toll and DmMyD88/Tube might either completely or partially abolish the recruitment and activation of Pelle to produce a range of dorsalized phenotypes. According to this model, active Toll interacts with a Spätzle dimer via its ectodomain and with a Wek dimer via its cytoplasmic tail. However, overexpression of Wek can not partially rescue the completely dorsalized phenotype of spz mutant embryos, indicating that Wek alone is not sufficient to mediate Toll activation (Chen, 2006).

Although the genetic and biochemical data support that the zinc finger-containing Wek acts at the plasma membrane as an adaptor during dorsoventral patterning, Wek preferentially accumulates in the nucleus of fat body cells. Thus, the possibility that Wek might have a nuclear function as well cannot be completely excluded. This would be reminiscent of Armadillo, which acts not only as an adaptor of Cadherin but also as a transcription factor in the nucleus (Chen, 2006).

Surprisingly, the data suggest that wek is not required for the Toll-mediated induction of the drosomycin gene in response to immune challenge. Indeed, wild-type responses were observed in the fat body of wek mutant flies, and in S2 cells depleted of wek mRNA by RNAi. This result might be explained by a different threshold level for wek function in development and immunity: the residual activity of the Toll pathway in wek^lor/wek^EX14 transheterozygote flies, and in wek dsRNA-treated S2 cells, could be sufficient for the immune response. It is noted, however, that experiments with DmMyD88 revealed on the contrary that the dorsoventral patterning function appears to be less sensitive than the immune function, as flies homozygote for the hypomorphic allele DmMyD88^EP2133 are severely impaired in their host-defense functions, but not for dorsoventral patterning. Thus, although a role of wek in Toll-mediated immune defenses cannot be formally rule out, the most likely explanation for the results is that induction of drosomycin expression by Toll in fat body and S2 cells does not depend on wek. This interpretation is supported by the fact that in the fat body Wek does not colocalize with Toll at the plasma membrane but rather preferentially localizes to the nucleus (Chen, 2006).

Differences between Toll signaling in the embryo and in adults have already been reported in several instances. The first difference pertains to the identity of the transcription factor induced, Dorsal in the embryo and Dif in adults. There is also convincing evidence that Toll not only signals to Cactus, but also induces phosphorylation of Dorsal. In addition, differences were observed in the interaction of Dorsal and Dif with cofactors. For example, unlike Dorsal, Dif does not appear to interact and synergize with basic-helix-loop-helix transcription factors or to be affected by the negative regulator WntD. Conversely, the coactivator dTRAP80 modulates activation of Dif, but not Dorsal, in S2 cells. Wek might therefore regulate a Dorsal-specific output of the Toll pathway. This hypothesis is, however, difficult to reconcile with the fact that Wek acts between two components of the pathway, Toll and DmMyD88, which are both required for activation of Dorsal in the embryo and Dif in adults. Furthermore, the proposed function of Wek as an adaptor that connects Toll and DmMyD88 does not explain why this factor is not required in adults and S2 cells. The most likely reason to explain this paradox is that another molecule substitutes for Wek in fat body and S2 cells. The three paralogs of wek (CG6254, CG17568, and CG10366) found in the Drosophila genome are prime candidates to carry this function and connect Toll to DmMyD88 in immune-competent cells. The first two contain both the N region and the zinc finger-containing C region, whereas the third one contains all three domains. However, RNAi-mediated silencing of these genes in S2 cells did not affect Toll signaling. Thus, the identity of the factor that bridges Toll and DmMyD88 in fat body cells remains unknown at this stage (Chen, 2006).

The different requirement for Wek in embryos and adults may also reflect the presence of Dorsal and Dif in the Toll receptor complex. Because the NF-κB-like molecules targeted by Toll are different in the embryo and in adults, such a mechanism could provide an explanation for the embryo-specific phenotype of wek mutant flies. In support of this hypothesis, Cactus-bound Dorsal has been shown to form a complex with Tube and Pelle, in which Cactus may be phosphorylated by Pelle. It is interesting to note that this hypothetic model of activation of Dorsal and Dif at the receptor complex is evocative of the activation of the transcription factor IRF7 by the kinase IRAK1 upon stimulation of TLR7 or TLR9 in mammalian cells (Chen, 2006).

GENE STRUCTURE

Bases in 5' UTR - 192

Bases in 3' UTR - 431

PROTEIN STRUCTURE

Amino Acids - 462

Structural Domains

Deletion analysis, together with an evolutionary comparison of tube genes in Drosophila species has defined two domains for the Tube protein. The amino-terminal domain is well conserved and is sufficient to rescue tube null embryos. The C-terminal half of Tube protein contains five copies of an eight-residue motif and shares no significant sequence similarity with known proteins (Letsou, 1991 and Letsou, 1993).

The interaction between the death domains (DDs) of Tube and the protein kinase Pelle is an important component of the Toll pathway. Published crystallographic data suggests that the Pelle-Tube DD interface is plastic and implies that in addition to the two predominant Pelle-Tube interfaces, a third interaction is possible. The NMR solution structure of the isolated death domain of Pelle is presented along with a study of the interaction between the DDs of Pelle and Tube. The data suggests the solution structure of the isolated Pelle DD is similar to that of Pelle DD in complex with Tube. Additionally, they suggest that the plasticity observed in the crystal structure may not be relevant in the functioning death domain complex (Moncrieffe, 2005).

The crystal structure suggests that the PelleÐTube DD complex can exist as a tetramer comprised of Pelle and Tube heterodimers arranged in a linear sequence P1:T1:P2:T2 and whether this persists in solution needs to be determined as it may have implications for the function of the DD complex. To address these issues, the structure of the isolated Pelle-DD in solution and the interaction between the DDs of Pelle and Tube have been solved using nuclear magnetic resonance (NMR) spectroscopy. The results suggest that in the beta-helical regions, the structure of Pelle-DD is similar to that of Pelle-DD in complex with the DD of Tube, and of the two types of Pelle-Tube dimer interfaces observed in the crystal structure (P1:T1 and P2:T2) only one is likely to persist in solution (Moncrieffe, 2005).

The NMR data presented confirm the extensive nature of the Pelle-Tube death domain interface because all twenty seven residues that constitute the core interaction surface on Pelle-DD show significant chemical shift differences between the ¹H-¹⁵N HSQC spectrum of Pelle-DD and the ¹H-¹⁵N HSQC-TROSY spectrum of the Pelle-Tube DD complex. The NMR data also suggest that the Tube1:Pelle2 interaction observed in the crystals, may persist in solution. This implies that the death domains of Pelle and Tube are capable of forming a tetramer and this is corroborated by the sedimentation velocity data which reveals that the concentration of the tetrameric complex is very small (0.4%) relative to that of the dimer. The dominant species formed by the interaction of Pelle-DD and Tube-DD is a dimer and whether the very small amount of the tetrameric species plays a role in signaling is unknown; for example, a mutant of Pelle bearing a mutation in the Tube1:Pelle2 interface (D50K) fails to express protein. It is conceivable that the residues in Pelle that are part of the Tube1:Pelle2 interface are sites of interaction for other members of the signaling complex. A recent report argues against that partner being dMyD88 since this component is thought to bind predominantly to Tube. A more likely scenario however, given that dMyD88 appears to bind predominantly to Tube and not Pelle, is that in the functioning complex, P2 is occupied by dMyD88. Thus, the residues in Tube-DD that contribute to the T1:P2 interface are likely to be the scaffold that recruits dMyD88 (Moncrieffe, 2005).

In summary, the structure of the isolated Pelle DD is similar to that of the Pelle DD in complex with the DD of Tube. While the death domains of Pelle and Tube may form a tetramer, consistent with the crystallographic observation the concentration of this species is extremely low. Consequently, inferences regarding the “plasticity” of the Pelle-Tube DD complex in vivo should be reevaluated (Moncrieffe, 2005).

date revised: 12 June 2021

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