InteractiveFly: GeneBrief

approximated: Biological Overview | References

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, app^e6 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. app^e6 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 app^e6 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, app^e1, app^e3, and app^e6 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. app^e3 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. app^e6 and app^e1 contain nonsense mutations predicted to truncate the protein prior to or toward the end of the DHHC-CRD, respectively, and therefore app^e6 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 app^e6 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 app^e6 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 app^e6 clones did not affect PCP, whereas large clones only affected PCP in the regions of the wing where defects were observed in app^e6 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. ft^fd and ft^G-rv are likely null alleles predicted to truncate Ft N-terminal to its transmembrane region. ft^fd homozygotes and ft^fd/ft^G-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 ft^fd; app^e6 and ft^fd/ft^G-rv; app^e6 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 app^e6. Null d^GC13 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 app^e6 clones, d^GC13 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 app^e6 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 app^e6 mutants and partly rescued the crossvein spacing and leg-joint defects of app^e6. 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 app^e6 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 app^e6 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).

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 Citation: 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 Citation: 15342474

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

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

Lobo, S., et al. (2002). Identification of a Ras palmitoyltransferase in Saccharomyces cerevisiae. J. Biol. Chem. 277: 41268-41273. PubMed Citation: 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 Citation: 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 Citation: 18804377

Nadolski, M. J. and Linder, M. E. (2007). Protein lipidation. FEBS J. 274: 5202-5210. PubMed Citation: 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 Citation: 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 Citation: 15632165

Roth, A. F., et al. (2006). Global analysis of protein palmitoylation in yeast. Cell 125: 1003-1013. PubMed Citation: 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 Citation: 16000296

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

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 Citation: 12379641

date revised: 20 July 2009

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