InteractiveFly: GeneBrief

approximated: Biological Overview | References

Gene name - approximated

Synonyms -

Cytological map position - 69A3-69A4

Function - enzyme

Keywords - DHHC family, palmitoylation, Fat signaling, planar cell polarity

Symbol - app

FlyBase ID: FBgn0260941

Genetic map position - 3L:12,199,338..12,259,889 [-]

Classification - DHHC zinc finger domain

Cellular location - surface transmembrand

NCBI link: EntrezGene

app orthologs: Biolitmine

Signaling via the large protocadherin Fat (Ft), regulated in part by its binding partner Dachsous (Ds) and the Golgi-resident kinase Four-jointed (Fj), is required for a variety of developmental functions in Drosophila. Ft and, to a lesser extent, Ds suppress overgrowth of the imaginal discs from which appendages develop and regulate the Hippo pathway. Ft, Ds, and Fj are also required for normal planar cell polarity (PCP) in the wing, abdomen, and eye and for the normal patterning of appendages, including the spacing of crossveins in the wing and the segmentation of the leg tarsus. Ft signaling has been shown to be negatively regulated by the atypical myosin Dachs (Cho, 2004; Mao, 2006). This study identifies an additional negative regulator of Ft signaling in growth control, PCP, and appendage patterning, the Approximated (App) protein. App encodes a member of the DHHC family, responsible for the palmitoylation of selected cytoplasmic proteins. Evidence is provided that App acts by controlling the normal subcellular localization and activity of Dachs (Matakatsu, 2008).

Crossvein spacing and tarsal leg segmentation are extremely sensitive to changes in Fat (Ft) activity; they are disrupted in weak Ft-pathway mutants that have no obvious growth or planar cell polarity (PCP) defects. Similar disruption occurs in app1 homozygotes: The distance between the anterior crossvein (ACV) and posterior crossvein (PCV) is reduced, and one or more tarsal leg joints are lost or reduced. app1 hemizygote wings also have weak PCP defects. Ethyl methanesulfonate was used to generate additional mutations that failed to complement app1. Of these, appe6 was semilethal in homozygotes and hemizygotes, and escaper adults had more extensive wing PCP defects, both proximally and in a distal region between the third and fourth longitudinal veins. They also had abdominal PCP defects: In the anterior compartment, most hairs point in the normal posterior direction, but polarity was disturbed around the anterior-posterior (A/P) boundary and extensively reversed in the posterior compartment. appe6 appears amorphic, given that the defects were not noticeably stronger in hemizygotes (Matakatsu, 2008).

The development of PCP in the pupal wing is accompanied by the polarized redistribution of the 'core' planar-polarity proteins to the proximal, distal, or proximal and distal faces of single wing cells. PCP mutants can be separated according to their effects on this polarization. Mutations in the core PCP proteins reduce the levels and block the polarization of the other core PCP proteins, whereas changes in ft, ds, or fj expression can reorient core PCP protein polarization along inappropriate axes. The levels of the core PCP protein Flamingo (Fmi, also known as Starry night) were not reduced in appe6 mutant clones (more than 30 examined), and Fmi polarization was in some cases reoriented. This further supports App's involvement in Ft signaling (Matakatsu, 2008).

app was mapped to a portion of 69A2-A4 containing seven known or predicted genes. app1, appe1, appe3, and appe6 all contained mutations in the 5' coding exons of one of these, the CG5620 Flybase gene prediction. A UAS-driven RNAi transgene was constructed corresponding to the 5' end of CG5620; it was expressed in developing dorsal wings by using ap-gal4; this produced app-like wing and PCP defects on the dorsal surface (Matakatsu, 2008).

However, the 3' end of the CG5620 coding prediction is in error. Products obtained by using 3' RACE contained instead the 3' exon of the adjacent CG17144 prediction, as did a BDGP EST. This transcript was called app-RA, and it was shown that the corresponding App-PA protein is produced in flies. Another EST predicts a shorter transcript called app-RB. No transcripts were found covering the entire final coding exon of CG5620 in embryonic or larval cDNA libraries or by RACE. However, the full CG5620 prediction is conserved in Drosophila pseudoobscura, suggesting that it might be utilized; this was termed app-RC (Matakatsu, 2008).

The wing and leg defects of app homozygotes were rescued by expressing UAS-app-RA, UAS-app-RB, or UAS-app-RC with either act-gal4 or en-gal4. Overexpression of higher levels of UAS-app with strong drivers such as ap-gal4 or tub-gal4 also disrupted PCP in the proximal wing and abdomen (Matakatsu, 2008).

app encodes a member of the DHHC protein family responsible for adding palmitates to cytoplasmic proteins (Linder, 2007; Nadolski, 2007). Eukaryotes contain multiple members of the DHHC family, with 8 predicted in yeast, 23 in mammals (Fukata, 2004), and 20 in Drosophila. The region common to all predicted App isoforms contains four predicted transmembrane domains, with a DHHC cysteine-rich domain (DHHC-CRD) located between the second and third transmembrane domains. It is likely that the DHHC-CRD is located on the cytoplasmic side of the membrane, as in the yeast DHHC protein AKR1 (Politis, 2005; Matakatsu, 2008 and references therein).

Alignments using the region common to the App isoforms indicate that App is in the same subfamily as the human ZDHHCs 9, 14, and 18 (Fukata, 2004) and is the Drosophila protein most similar to yeast ERF2 (Swarthout, 2005). The similarity between App and these proteins drops off at the C-terminal end of the common region. The divergent C termini of App-PA and App-PC have no significant similarity to each other or to other proteins in the database outside the drosophilids, except for a short region at the C terminus of App-PA that is similar to predicted App homologs from the insects Tribolium and Apis. App-PB has a much shorter C terminus. Because any of the putative app transcripts rescued the app leg and wing phenotypes, the different C termini are dispensable for these phenotypes (Matakatsu, 2008).

app1 contains a missense mutation N-terminal to the DHHC-CRD domain and an aberration that introduces a frameshift predicted to truncate the C-terminal end of App-PA. appe3 contains a missense mutation that changes a conserved cysteine in the DHHC-CRD. Because the DHHC-CRD is required for palmitoyltransferase activity (Lobo, 2002), this supports a role for palmitoylation in Ft signaling. appe6 and appe1 contain nonsense mutations predicted to truncate the protein prior to or toward the end of the DHHC-CRD, respectively, and therefore appe6 is likely null for App function (Matakatsu, 2008).

Two antisera were generated, one specific for App-PA and one for the common region. Both antisera uniformly stained embryos, imaginal discs, and pupal wings, and staining was lost from mitotic homozygous appe6 clones, confirming the expression of the App-PA isoform; similar results with anti-App-common. There is no obvious asymmetric distribution of the App protein along the proximodistal or anterior-posterior axes of imaginal discs or pupal wings. However, staining was especially strong in the apical cell cortex, and this concentration did not extend more basally to the adherens junction marker DE-cadherin. This is similar to the distribution of Ft and Ds, and there is overlap between the regions where App, Ds, and Ft are concentrated. This result is surprising because human ZDHHC9, 14, and 18 and yeast ERF2 are concentrated in the Golgi or ER; only a few, less similar ZDHHCs have been detected at the plasma membrane (Swarthout, 2005; Bartels, 1999; Ohno; 2006; Zhao, 2002). No significant overlap was observed between App and Golgi or ER markers in wing discs. Although App must traffic through the ER and Golgi, these results suggest that App is active in the plasma membrane, in or near the apical region where Ft and Ds are concentrated (Matakatsu, 2008).

However, App does not detectably regulate Ft and Ds levels or their binding. The binding between Ft and Ds stabilizes both proteins at the cell surface in wing discs, but appe6 clones did not affect Ft or Ds levels or distribution. Creating artificial boundaries of ft or ds expression also strongly polarizes wing PCP, as do boundaries of the Fj kinase that phosphorylates Ft and Ds and modulates their levels. If App affected Ft-Ds levels or binding, App boundaries would be expected to affect PCP. However, small to moderately sized appe6 clones did not affect PCP, whereas large clones only affected PCP in the regions of the wing where defects were observed in appe6 homozygotes. There was no tendency to reorient hairs at clone boundaries, and only rarely did regions with altered PCP affect PCP in adjacent wild-type cells; these findings may be due to altered cell interactions mediated by the core polarity proteins. Sharp boundaries of UAS-app-RA misexpression (e.g., driven with the posterior driver en-gal4) also had no effect on PCP. Anti-App staining was not altered in wing discs by ft or ds clones. Thus, despite their colocalization, there is no evidence that App physically interacts for Ft or Ds (Matakatsu, 2008).

PCP defects, reduced crossvein spacing, and lost tarsal leg joints can be caused by either gains or losses in Ft signaling, so the phenotypes of app ft double mutants were examined. ftfd and ftG-rv are likely null alleles predicted to truncate Ft N-terminal to its transmembrane region. ftfd homozygotes and ftfd/ftG-rv die during late pupal stages with strongly overgrown imaginal discs and disc-derived tissues; late pupal abdomens are not overgrown but have strong PCP defects. These phenotypes were partially rescued in ftfd; appe6 and ftfd/ftG-rv; appe6 flies: Overgrowth and extra folding of imaginal discs were suppressed and lethality and abdominal PCP defects were reduced. PCP was still defective in proximal portions of the wing but was normal in the distal wing, in contrast to the distal defects in viable ft18 wings. Thus, App acts genetically downstream of and in opposition to Ft in both growth control and PCP (Matakatsu, 2008).

The effects of app mutations on Ft signaling are strikingly similar to those caused by reducing the function of the atypical myosin Dachs (Mao, 2006). Like app mutations, dachs (d) mutations partially suppress the overgrowth and PCP defects caused by loss of Ft (Cho, 2004; Mao, 2006). The adult phenotypes are also similar, although the defects in d null mutants are stronger than those of appe6. Null dGC13 hemizygotes and hypomorphic d1 homozygotes reduce tarsal leg segmentation and the distance between the ACV and PCV and cause mild wing PCP defects that are quite similar to those observed in null app mutants. Like appe6 clones, dGC13 clones had PCP defects when in regions of the wing where defects occur in d homozygotes, and Fmi polarization was reoriented in d1 pupal wings. d mutants also had abdominal PCP defects similar to those in app mutants: Polarity was almost normal in the anterior compartment, but abnormal near the A/P compartment boundary and reversed in the posterior compartment (Matakatsu, 2008).

Therefore the effects of App on the levels and distribution of a V5-tagged Dachs protein were examined. Dachs:V5 normally accumulates at higher levels in the apical cell cortex in wing imaginal discs (Mao, 2006), overlapping the region of high anti-App staining. Apical Dachs accumulation was greatly reduced, although not completely eliminated, in appe6 clones. No changes were detected in the levels of basolateral or cytoplasmic Dachs:V5. Although App affects Dachs accumulation at the apical cell cortex, anti-App staining was normal in d mutant clones (Matakatsu, 2008).

Co-overexpression of App and Dachs:V5 greatly increased the accumulation of Dachs at the cell cortex compared with the expression of Dachs:V5 alone. Coexpression of App also increased the efficacy of Dachs in growth and PCP. Even though overexpression of App-RA did not obviously increase growth, coexpression of App-RA and Dachs caused greater overgrowth than did the expression of Dachs alone. Co-overexpression of Dachs and App caused more extreme PCP defects in the wing and abdomen than did the overexpression of App alone, whereas overexpression of Dachs:V5 alone did not affect PCP (Matakatsu, 2008).

It is likely that much or all of the app mutant phenotype is mediated by the reduction of effective Dachs at the apical cell cortex. The effects of app and d mutants are not additive: Double mutants for null app and d alleles resembled the stronger d null phenotype, as expected if App works by controlling Dachs activity. Because App affects Dachs post-transcriptionally, it is unlikely that overexpressed Dachs would fully rescue the app null. Nonetheless, overexpression of UAS-d with ap-gal4 or en-gal4 rescued the wing PCP defects normally found in the distal wing of appe6 mutants and partly rescued the crossvein spacing and leg-joint defects of appe6. That Dachs retains some activity in the absence of App is consistent with the low but significant levels of Dachs that remain at the apical cell cortex in appe6 clones. Different DHHC proteins can palmitoylate the same target (Roth, 2006), so other Drosophila DHHC proteins may be supplying residual activity in the absence of App (Matakatsu, 2008).

It is concluded that App palmitoyltransferase acts in opposition to the Ft pathway, largely or wholly by controlling the apical-cell-cortex localization and the activity of the atypical myosin Dachs. This localization is probably required for full Dachs activity. For growth control, this localization would place Dachs near not only Ft, but also the Hippo pathway member Warts. Warts is concentrated near the cell cortex, with an apical bias that overlaps the region of strong App and Dachs accumulation. Dachs binds Warts and may thereby regulate the Hippo pathway, accounting for its effects on Ft-dependent growth control (Matakatsu, 2008).

Dachs also modulates the effects of Ft signaling on PCP. It is not clear whether this modulation is also mediated through changes in Warts activity. Warts is thought to act by changing the activity of the transcription factor Yorkie, an effect that would not directly confer polarity. Moreover, Dachs was reported to accumulate preferentially on the distal faces of some wing-disc cells (Mao, 2006), suggesting that Dachs is involved in cell polarization independent on any effect on transcription, probably via as-yet-unknown binding partners (Matakatsu, 2008).

It remains possible that Dachs is palmitoylated by App. However, there is no precedent for palmitoylation of a myosin, nor does Dachs score highly when an algorithm is used that detects palmitoylation sites. Nor has Dachs palmitoylation been detected by using the acylbiotin-exchange technique. The effect of App may thus be less direct, palmitoylating a binding partner or regulator of Dachs. Although Dachs is a myosin, portions of Dachs are unique and lack known protein-interaction motifs (Mao, 2006; Tzolovsky, 2002). Warts is the only proven binding partner for Dachs, but appe6 clones did not affect the levels or cell-cortex localization of Myc-tagged Warts in wing discs (Matakatsu, 2008).

Given that the human and yeast DHHC proteins that App most resembles palmitoylate many targets (Swarthout, 2005; Lobo, 2002; Roth, 2006), the adult phenotypes of app mutants are surprisingly specific to the Ft pathway. One known target of ERF2 and ZDHHC9 is Ras, whose activity relies on membrane localization through both farnesylation and palmitoylation. Intriguingly, the Ras and MAPK pathways interact with the Ft pathway in growth control. However, reducing Ras activity causes loss of wing veins, a phenotype not observed in app mutations, and does not cause the PCP and appendage-patterning defects of app and d mutants. Moreover, reducing Ras activity via expression of a dominant negative EGF receptor did not affect the levels of Dachs:V5 in wing discs. The different subcellular distributions of App, to the cell cortex, and ZDHHC9 and ERF2, to endomembranes, suggests they have different roles and targets, despite their strong similarity at the amino acid level (Matakatsu, 2008).

The novel SH3 domain protein Dlish/CG10933 mediates fat signaling in Drosophila by binding and regulating Dach

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 (Dachs ligand with SH3s). 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).


Search PubMed for articles about Drosophila Approximated

Bartels, D. J., et al. (1999). Erf2, a novel gene product that affects the localization and palmitoylation of Ras2 in Saccharomyces cerevisiae. Mol. Cell. Biol. 19: 6775-6787. PubMed ID: 10490616

Cho, E. and Irvine, K. D. (2004). Action of fat, four-jointed, dachsous and dachs in distal-to-proximal wing signaling. Development 131: 4489-4500. PubMed ID: 15342474

Fukata, M., et al. (2004). Identification of PSD-95 palmitoylating enzymes, Neuron 44: 987-996. PubMed ID: 15603741

Linder, M. E. and Deschenes, R. J. (2007). Palmitoylation: Policing protein stability and traffic. Nat. Rev. Mol. Cell Biol. 8: 74-84. PubMed ID: 17183362

Lobo, S., et al. (2002). Identification of a Ras palmitoyltransferase in Saccharomyces cerevisiae. J. Biol. Chem. 277: 41268-41273. PubMed ID: 12193598

Mao, Y., et al. (2006). Dachs: An unconventional myosin that functions downstream of Fat to regulate growth, affinity and gene expression in Drosophila. Development 133: 2539-2551. PubMed ID: 16735478

Matakatsu, H. and Blair, S. S. (2008). The DHHC palmitoyltransferase approximated regulates Fat signaling and Dachs localization and activity. Curr. Biol. 18(18): 1390-5. PubMed ID: 18804377

Nadolski, M. J. and Linder, M. E. (2007). Protein lipidation. FEBS J. 274: 5202-5210. PubMed ID: 17892486

Ohno, Y., Kihara, A., Sano, T. and Igarashi, Y. (2006). Intracellular localization and tissue-specific distribution of human and yeast DHHC cysteine-rich domain-containing proteins. Biochim. Biophys. Acta 1761: 474-483. PubMed ID: 16647879

Politis, E. G., Roth, A. F. and Davis, N. G. (2005). Transmembrane topology of the protein palmitoyl transferase Akr1. J. Biol. Chem. 280: 10156-10163. PubMed ID: 15632165

Roth, A. F., et al. (2006). Global analysis of protein palmitoylation in yeast. Cell 125: 1003-1013. PubMed ID: 16751107

Swarthout, J. T., et al. (2005), DHHC9 and GCP16 constitute a human protein fatty acyltransferase with specificity for H- and N-Ras. J. Biol. Chem. 280: 31141-31148. PubMed ID: 16000296

Tzolovsky, G., et al. (2002). Identification and phylogenetic analysis of Drosophila melanogaster myosins. Mol. Biol. Evol. 19: 1041-1052. PubMed ID: 12082124

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

Zhao, L., et al. (2002). Erf4p and Erf2p form an endoplasmic reticulum-associated complex involved in the plasma membrane localization of yeast Ras proteins. J. Biol. Chem. 277: 49352-49359. PubMed ID: 12379641

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