InteractiveFly: GeneBrief

Dachs ligand with SH3s: Biological Overview | References

Much of the Hippo and planar cell polarity (PCP) signaling mediated by the Drosophila protocadherin Fat depends on its ability to change the subcellular localization, levels and activity of the unconventional myosin Dachs. To better understand this process, a structure-function analysis of Dachs was performed, and this was used to identify a novel and important mediator of Fat and Dachs activities, a Dachs-binding SH3 protein that has been named Dlish. Dlish was found to be regulated by Fat and Dachs. Dlish also binds Fat and the Dachs regulator Approximated, and Dlish is required for Dachs localization, levels and activity in both wild type and fat mutant tissue. The evidence supports dual roles for Dlish. Dlish tethers Dachs to the subapical cell cortex, an effect partly mediated by the palmitoyltransferase Approximated under the control of Fat. Conversely, Dlish promotes the Fat-mediated degradation of Dachs (Zhang, 2016).

Heterophilic binding between the giant Drosophila protocadherins Fat and Dachsous (Ds) both limits organ growth, via regulation of the Hippo pathway, and orients planar cell polarity (PCP), through cell-by-cell polarization of Fat, Ds and their downstream effectors. Loss of Fat and, to a lesser extent, Ds, leads to the profound overgrowth of the Drosophila imaginal discs that give rise to adult appendages, and loss of either disorders the polarity of cell divisions, hairs and other morphological features in a variety of Drosophila tissues. But while players and pathways have been defined that are genetically downstream of Fat-Ds binding, only a little is known about the biochemical links between these and their most powerful regulator, the intracellular domain (ICD) of Fat (Zhang, 2016).

A good deal of the recent work on Fat effectors has focused on the regulation of unconventional type XX myosin Dachs. Dachs is critical first because it provides the only known marker specifically sensitive to changes in the Fat/Ds branches of both the Hippo and PCP pathways. Dachs is normally concentrated in the subapical cell cortex, overlapping subapically-concentrated Fat and Ds. Loss of Fat greatly increases subapical Dachs levels, and polarization of Fat and Ds to opposite cell faces can in turn polarize Dachs to the face with less Fat. Fat thus inhibits or destabilizes subapical Dachs, while Ds may do the opposite. Downstream changes in Hippo or PCP activities do not affect Dachs (Zhang, 2016).

Dachs changes are also critical because they play a major role downstream of Fat. Dachs binds to and inhibits the activity of the kinase Warts (the Drosophila Lats1/2 ortholog), both reducing Warts levels and changing its conformation. Warts is concentrated in the subapical cell cortex, and thus the increased cortical Dachs of fat mutants should reduce the phosphorylation of Yorkie by Warts, allowing Yorkie to move into the nucleus to drive the transcription of growth-promoting target genes. Indeed, Dachs is necessary for the overgrowth and increased Yorkie target gene expression of fat mutants. Dachs overexpression also causes overgrowth, although more weakly than the overgrowth caused by the loss of Fat, indicating that Dachs is partly sufficient (Zhang, 2016).

Dachs can also bind to the core PCP pathway component Spiny legs (Sple) and alter its localization, thus influencing PCP in the subset of tissues that rely on Sple. The increased levels of unpolarized Dachs in fat mutants may misdirect Sple, accounting for at least some of the PCP defects; fat mutant hair PCP defects are improved, although not eliminated, by loss of dachs (Zhang, 2016).

Dachs has not been shown to interact directly with Fat's ICD, and only three other proteins are known to affect Dachs accumulation in the subapical cell cortex, the casein kinase ε Discs overgrown (Dco), Approximated (App) and F-box-like 7 (Fbxl7). Dco may act through Fat itself: Dco binds and phosphorylates the Fat ICD, and loss of Dco function causes strong overgrowth and increases subapical Dachs, similar to loss of Fat (Zhang, 2016).

App suggests a mechanism in which Fat inhibits the tethering of Dachs to protein complexes in the subapical domain. app mutants decrease subapical Dachs levels and reduce Dachs activity. Thus, like dachs mutants, app mutants reverse the overgrowth and increased Yorkie target gene expression normally observed in fat mutants, and improve hair PCP. App is one of 20 Drosophila DHHC palmitoyltransferases, transmembrane proteins responsible for adding palmitates to cytoplasmic proteins and thereby anchoring them to cell membranes. App is also concentrated in the subapical cell membrane and can bind both Dachs and the Fat ICD. Thus, in the simplest model App palmitoylates or tethers Dachs, concentrating it in the cell cortex, and Fat works in part by sequestering or inhibiting App. However, Dachs is not detectably palmitoylated (Zhang, 2016).

This study describes the function of a novel Dachs binding protein, and shows that its effects provide strong evidence for both the palmitoylation-dependent and degradation-dependent regulation of Dachs. Structure-function analysis of Dachs found regions required for its normal subapical localization, and this information was used as the basis for a screen for novel Dachs binding partners. A direct binding partner was found for the Dachs C-terminus, the previously uncharacterized SH3 domain protein CG10933, which has been renamed Dachs ligand with SH3s, or Dlish. The activity and subapical concentration of Dlish are regulated by Fat, Dco and Dachs, and Dlish in turn is required for the subapical concentration and full activity of Dachs in both wild type and fat mutant cells. Dlish localization also depends on App; furthermore Dlish binds to and is palmitoylated by App, and palmitoylation can be suppressed by Fat. Loss of Dlish also increases the total levels of Dachs, likely by blocking Fat-mediated destabilization of Dachs. It is proposed that Dlish targets Dachs to subapical protein complexes in part via Fat-regulated, App-mediated palmitoylation. Dlish thereby concentrates Dachs where it can efficiently inhibit subapical Warts, and conversely links Dachs to the machinery for Fat-dependent destabilization (Zhang, 2016).

The unconventional myosin Dachs is an important effector Fat/Ds-regulated Hippo signaling, as its heightened subapical levels in fat mutants inhibit and destabilize Warts, freeing Yorkie to increase the expression of growth-promoting genes. A structure-function analysis of Dachs was used as a springboard to search for new binding partners that are critical for Dachs localization and function, and have found Dlish (CG10933), a novel SH3 domain protein. Dlish binds directly to the Dachs C-terminus; loss of Dlish disrupts Dachs localization, levels and function: subapical accumulation of Dachs is reduced and cytoplasmic and total levels increase, both in wild type and fat mutant tissue, while activity is lost. Importantly, Dlish is regulated by Fat, as loss of Fat greatly increases Dlish levels in the subapical cell cortex and, like Dachs, Dlish is needed for much of the fat mutant overgrowth (Zhang, 2016).

Dlish also binds the ICD of Fat and other Fat-binding proteins, including two that likely mediate part of its function: the palmitoyltransferase App and the F-box protein Fbxl7. Thus Dlish provides a new biochemical link from the Fat ICD to Dachs regulation. Evidence indicates that Dlish plays two different and opposing roles (see Models of Fat-mediated regulation of subapical Dachs by Fat-inhibited subapical tethering and Fat-stimulated destabilization). First, it helps tether Dachs in the subapical cell membrane, in part via Fat-regulated, App-dependent palmitoylation, so that Dachs can more efficiently inhibit Warts. Second, it links Dachs to Fat-organized machinery for Dachs destabilization, including Fbxl7, and thus helps reduce Dachs levels (Zhang, 2016).

Dlish and Dachs cooperate to target or tether the Dlish-Dachs complex, as each is necessary, and to a weaker extent sufficient, for the subapical concentration of the other. The Dachs contribution is likely through tethering the complex to the cortical cytoskeleton, as this study found that loss of the F-Actin-binding myosin head blocks the subapical localization of Dachs. This would agree with recent biochemical analyses that suggest that Dachs has no motor function, acting rather as an F-Actin-binding scaffolding protein (Zhang, 2016).

The Dlish contribution, on the other hand, depends at least in part on its ability to bind the transmembrane DHHC palmitoyltransferase App. Loss of App and Dlish have very similar effects on Dachs localization and activity. This study found that loss of App disrupts the subapical accumulation of Dlish in vivo and that App can stimulate palmitoylation of Dlish in vitro. Thus, palmitoylation of Dlish likely stimulates membrane association of both Dlish and its binding partner Dachs (Zhang, 2016).

App also has additional effects on Fat pathway activity. First, App has palmitoyltransferase-independent activity and can co-IP Dachs in vitro. Thus, while palmitoylation of Dlish may mediate some of App's activity, subapical App may simultaneously help localize the Dlish-Dachs complex by physical tethering. And while both palmitoylation and tethering of the Dlish-Dachs complex is likely critical for the fat mutant phenotype, App also has a function that depends on the presence of Fat, as App can bind, palmitoylate and inhibit the activity of Fat's ICD (Zhang, 2016).

An important question is whether the absence of Fat regulates the App-dependent tethering of the Dlish-Dachs complex. The Fat ICD can complex with both Dlish and App. Dlish and App can bind not only the C-terminal region of the Fat ICD where Fat is palmitoylated, but also the PH and Hippo domains which was shown to played the strongest role in Dachs regulation. An attractive mechanism is that Fat inhibits the interaction between App and Dlish, reducing App's ability to palmitoylate and tether the Dlish-Dachs complex. In the absence of Fat, App and Dlish are freed to tether Dachs, and Dachs now inhibits and destabilizes Warts, causing overgrowth. In support of this model, it was found that overexpression of Fat's ICD in vitro can reduce App-stimulated palmitoylation of Dlish (Zhang, 2016).

The evidence further indicates that Dlish targets Dachs for Fat-dependent destabilization. Loss of Fat increases not only subapical Dachs, but also total Dachs levels. In the presence of Dlish the increased Dachs remains subapical. Loss of Dlish also increases the total levels of Dachs, but now that increase is cytoplasmic, and much less effective at inhibiting Warts. These Dachs increases are unlikely to have independent causes, as they are not additive; total Dachs levels are similar after loss of Fat, Dlish or both (Zhang, 2016).

It is proposed that in wild type cells there is a flux of the Dachs-Dlish complex from the cytoplasm to the subapical cell cortex, where a Fat-dependent complex destabilizes Dachs. Normally the tethering effects of Dlish predominate over the Fat-dependent destabilization, and moderate levels of subapical Dachs are maintained. Destabilization is lost without Fat; this combines with Dlish-mediated tethering to increase subapical Dachs. Without Dlish the subapical tethering of Dachs is disrupted, access to Fat-dependent destabilization is lost and the now cytoplasmic Dachs increases. The model thus explains why the excess, largely cytoplasmic Dachs caused by reduced Dlish function is not greatly influenced by the presence or absence of Fat (Zhang, 2016).

In addition to any effects caused by changing the subcellular localization of Dachs, Dlish may also provide a direct link to the machinery for protein ubiquitination, as Dlish can co-IP with the E3 ubiquitin ligase Fbxl7, as well as the related F-box ubiquitin ligase Slimb. Fbxl7 is particularly intriguing, as it binds to and is regulated by Fat's ICD, and reduces subapical Dachs, perhaps via ubiquitination. Slimb can bind and ubiquitinate Expanded, a subapical regulator of Hippo signaling with links to Fat and Dachs function. But while it was found that loss of Fbxl7 or Slimb increases subapical Dachs and Dlish, these effects are weak, and the large increase in total Dachs levels caused by loss of Dlish or Fat must involve additional partners (Zhang, 2016).

Mutations in Fat’s closest mammalian homolog Fat4 (FatJ) and its Ds-like ligands strongly disrupt PCP-like processes, and have in humans been associated with the multisystem defects of Hennekam and Van Maldergem syndromes. There has been some debate, however, about whether the mammalian proteins retain direct regulation of Hippo activity. Nonetheless, Fat4 has been linked to Hippo changes in both normal development and tumors, mutations in Fat4 or Dachsous1 change the balance of precursors and mature neurons in the developing neuroepithelium of both humans and mice, and the mouse defect can be reversed by knockdown of the Yki homolog Yap. But the mechanisms underlying these effects are unknown, and Fat4 cannot regulate Hippo signaling in Drosophila (Zhang, 2016).

It is therefore important to note that while homologs of Dachs and Dlish are found throughout the animal kingdom, they are apparently absent from vertebrates. This suggests that the Dachs-Dlish branch of the Fat-Ds pathway, with its powerful effect on Warts activity, is also lacking. Nonetheless, it has been suggested that Drosophila Fat and Ds can affect Hippo pathway activity in a Dachs-independent manner. It is also clear that Drosophila Fat has Dachs-independent effects on PCP; indeed the N-terminal 'PCP' domain of the Fat ICD that did not affect Dachs in this study is sufficient to improve the PCP defects of fat mutants. These or alternative pathways may still be present in mammals (Zhang, 2016).

Vamana couples fat signaling to the Hippo pathway

The protocadherins Dachsous and Fat initiate a signaling pathway that controls growth and planar cell polarity by regulating the membrane localization of the atypical myosin Dachs. How Dachs is regulated by Fat signaling has remained unclear. This study identified the vamana gene (CG10933) as playing a crucial role in regulating membrane localization of Dachs and in linking Fat and Dachsous to Dachs regulation. Vamana, an SH3-domain-containing protein, physically associates with and co-localizes with Dachs and promotes its membrane localization. Vamana also associates with the Dachsous intracellular domain and with a region of the Fat intracellular domain that is essential for controlling Hippo signaling and levels of Dachs. Epistasis experiments, structure-function analysis, and physical interaction experiments argue that Fat negatively regulates Dachs in a Vamana-dependent process. These findings establish Vamana as a crucial component of the Dachsous-Fat pathway that transmits Fat signaling by regulating Dachs (Misra, 2016).

Coordinated growth and morphogenesis is critical to the development of tissues of specific size and shape. Dachsous (Ds)-Fat signaling (henceforth, Fat signaling) controls both growth, through regulation of Hippo signaling, and morphogenesis, through regulation of planar cell polarity (PCP). Fat signaling regulates Hippo signaling and PCP by controlling the membrane localization of the atypical myosin protein Dachs. Many studies have provided important insights into both how Dachs influences Hippo signaling, and how it influences PCP. In contrast, the mechanism by which Fat signaling actually controls Dachs has remained less well understood (Misra, 2016).

Fat and Ds are atypical cadherins with novel intracellular domains (ICD), which localize to the plasma membrane just apical to the adherens junctions. Fat and Ds bind to each other in a heterophilic manner, and this interaction is modulated by the Golgi-resident kinase, Four-jointed (Fj), which phosphorylates their extracellular domains. This heterophilic binding, together with the graded expression of Ds and Fj, contribute to polarization of Ds and Fat localization within cells. Three different ways by which Fat signaling influences Hippo signaling have been described: Fat signaling influences the membrane localization of Expanded (Ex) , the levels of Wts protein, and the interaction of Wts with its cofactor Mats. Each of these effects on Hippo signaling depends upon Dachs. Fat signaling affects PCP in at least two ways: through an influence on junctional tension, and by regulating the Spiny-legs (Sple) isoform of the prickle locus. Both of these effects also involve Dachs (Misra, 2016).

Dachs was identified as a key downstream effector of Fat signaling because mutations in dachs completely suppress the overgrowth induced by fat mutations, and partially suppress the PCP defects induced by fat mutations. Dachs localizes to the cell membrane just apical to the adherens junction in a polarized manner; in the developing wing Dachs is localized to the distal sides of the cell, in response to the proximal-distal gradients of Ds and Fj expression. Dachs membrane localization requires a palmitoyltransferase encoded by approximated (app), but how App influences Dachs localization is unknown. In fat or ds mutants increased levels of Dachs are observed at the apical membrane and Dachs is no longer polarized. Forcing Dachs membrane localization by fusing it to Zyxin phenocopies fat mutants. Conversely, overexpression of full-length Fat or even just the Fat intracellular domain (ICD) displaces Dachs from the membrane into the cytoplasm. These and other observations have indicated that Fat regulates growth by modulating the levels of Dachs at apical membranes, and regulates Dachs-dependent PCP by directing Dachs asymmetry (Misra, 2016).

To understand how Fat functions, several studies have examined the roles of different regions of the Fat ICD. These studies identified two regions that mediate its growth-suppressive function. One, the D region, around amino acids 4,975 to 4,993, makes a modest contribution to Hippo pathway regulation, as when this region is deleted flies are viable but their wings are approximately 30% larger than normal, and also rounder than normal. The D region is required for interaction with the ubiquitin ligase, Fbxl7, which reduces Dachs membrane levels, and mutation of which results in phenotypes similar to deletion of the D region. A second region, which has been referred to as HM, Hpo, or H2, is defined by observations that deletions within this region block the ability of Fat to activate Hippo signaling. Two alleles of fat, fat⁶¹ and fat^sum, have also been identified that harbor mutations within this region, and are associated with tissue overgrowth comparable with that caused by fat null mutations. However, the mechanism by which this region, which for simplicity is referred to as the H region, regulates the Hippo pathway, and whether it affects Dachs, are unknown (Misra, 2016).

This study reports the isolation and characterization of the Src homology 3 (SH3)-domain-containing protein encoded by vamana (vam). Loss of vam function decreases growth, whereas overexpression of vam promotes growth. These effects are mediated through regulation of the Hippo pathway, and vam functions genetically downstream of fat, as vam mutations can suppress both growth and PCP phenotypes of fat. Vam localizes to the apical region of epithelial cells in a polarized manner, co-localizing with Dachs, and is required for normal membrane localization of Dachs. Vam physically associates with the carboxy-terminal domain of Dachs and the ICDs of Ds and Fat, and is regulated by the H region of the Fat ICD. These observations identify Vam as a key mediator of signaling from Fat to Dachs (Misra, 2016).

These studies identified the C-terminal region of Dachs as sufficient to mediate its interaction with Vam. Interestingly, the original dachs allele, described almost a century ago by Bridges and Morgan (1919), is a hypomorphic allele associated with insertion of a blood transposon just upstream of the C-terminal region. Hence this allele likely encodes a truncated protein that lacks the Vam-interaction domain. Consistent with this inference, the vam null phenotype appears similar to the dachs¹ phenotype. Thus, a requirement for interaction with Vam can explain the basis for the original identification of dachs (Misra, 2016).

Vam is evolutionarily conserved among insects but with no close homologs in vertebrates. This is consistent with the fact that Dachs is also only found in insects, and the sequence of the H region is not conserved in vertebrate Fat genes. Nonetheless, Vam is structurally related to a broad family of SH2- and SH3-domain-containing proteins exemplified by CRK, Grb2, Myd88, and NCK. These proteins are referred to as signal-transducing adapter proteins and facilitate formation of protein complexes that play key roles in signal transduction. Vam is composed of just three SH3 domains; this domain organization is most similar to that of the NCK family of adapters, which contain three SH3 domains along with one SH2 domain. The finding that Vam uses both SH3-1 and SH3-3 to interact with Fat and Ds is also reminiscent of NCK family adapters, as they engage effectors using multiple SH3 domains. The Drosophila ortholog of NCK, dreadlocks (dock), interacts with cell-adhesion molecules encoded by hibris, kirre, roughest, and sticks and stones (sns) to regulate actin polymerization and growth cone migration, and functional redundancy of SH3 domains has been observed for dock. Multiple SH3 domains are also commonly observed in proteins involved in vesicular trafficking. The observation that in vam mutants Dachs accumulates in cytoplasmic puncta that could be vesicular structures suggests that Vam might influence the trafficking of Dachs (Misra, 2016).

Fat and Ds proteins are conserved in vertebrates, where they play important roles in controlling PCP, and have also been proposed to influence Hippo signaling. In the absence of a Dachs homolog, however, it has been unclear how downstream signaling is mediated in vertebrate Ds-Fat pathways. The discovery that Vam links Ds and Fat to downstream signaling raises the possibility that a different member of the signal-transducing adapter proteins could mediate downstream Ds-Fat signaling in vertebrates (Misra, 2016).

The H region of the Fat ICD plays a crucial role in Hippo pathway regulation. This analysis of Fat ICD truncations revealed that the H region inhibits Vam and Dachs membrane accumulation, the influence of Fat ICD deletions on Hippo signaling correlates with their influence on Vam and Dachs membrane localization, and the H region of Fat can associate with Vam. Together with observations that Vam associates with and regulates Dachs, these observations lead to the inferrence that the H region normally functions to promote Hippo signaling through its association with, and regulation of, Vam. Fat also influences growth and Dachs accumulation through a second region of the ICD, the D region, which interacts with Fbxl7. Because mutation of the D region, or mutations in Fbxl7, have weaker phenotypes than mutations in the H region, the H region appears to play the larger role in Dachs regulation, but nonetheless it is expected that both regions normally act in parallel to regulate membrane levels of Dachs and thus, ultimately, Hippo signaling (Misra, 2016).

The localization of Vam in different genotypes, together with its physical interactions, suggests models for how Vam regulates Dachs localization. Since Vam and Dachs are reciprocally required for each other's membrane localization, it is inferred that a complex between these two proteins is required for their stable localization to apical junctions, where Dachs regulates PCP (via interactions with Sple) and Hippo signaling (via interactions with Zyxin and Warts). The observations that Fat promotes removal of Vam and Dachs from the subapical membrane, associates with Vam, yet does not visibly co-localize with Vam at apical junctions, suggests that Fat normally removes Vam-Dachs complexes from the subapical membrane. One mechanism by which this might occur is through binding of Fat to Vam, followed by endocytosis of Fat-Vam-Dachs complexes. Alternatively, Fat binding might disrupt Vam-Dachs binding, as these proteins normally do not localize to the membrane in isolation (Misra, 2016).

It was also observed that Vam can interact with the Ds ICD, and that it does so through the same SH3 domains as it uses to interact with the Fat ICD. This suggests that these interactions are likely to be competitive. In this case, interaction of Vam with the Ds ICD could promote Vam and Dachs membrane localization by opposing the influence of Fat on Vam. For example, by competing with Fat for binding to Vam, Ds could prevent Fat from disrupting Vam-Dachs interactions, or promoting endocytosis of a Vam-Dachs complex. Consistent with this suggestion that the Ds ICD stabilizes Vam and Dachs at apical junctions, Vam, Ds, and Dachs normally all co-localize in puncta on the distal side of wing cells. The ability of Vam to associate with the ICDs of both Fat and Ds could thus provide a simple mechanism explaining how the ICD of Ds seems to promote Dachs membrane localization, whereas the ICD of Fat inhibits it (Misra, 2016).

Search PubMed for articles about Drosophila Dlish or Vamana

Misra, J.R. and Irvine, K.D. (2016). Vamana couples fat signaling to the Hippo pathway. Dev Cell 39(2):254-266. PubMed ID: 27746048

Zhang, Y., Wang, X., Matakatsu, H., Fehon, R. and Blair, S. S. (2016). The novel SH3 domain protein Dlish/CG10933 mediates fat signaling in Drosophila by binding and regulating Dachs. Elife 5. PubMed ID: 27692068

date revised: 26 October 2017

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