shaggy: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

Gene name - shaggy

Synonyms - zeste white 3

Cytological map position - 3B1

Function - signal transduction, enzyme

Keywords - segment polarity, wingless pathway, component of the ß-catenin destruction complex

Symbol - sgg

FlyBase ID:FBgn0003371

Genetic map position - 1-1.3

Classification - serine-threonine kinase - glycogen synthase kinase 3

Cellular location - cytoplasmic



NCBI link: Entrez Gene
sgg orthologs: Biolitmine
Recent literature
Trostnikov, M. V., Veselkina, E. R., Krementsova, A. V., Boldyrev, S. V., Roshina, N. V. and Pasyukova, E. G. (2020). Modulated Expression of the Protein Kinase GSK3 in Motor and Dopaminergic Neurons Increases Female Lifespan in Drosophila melanogaster. Front Genet 11: 668. PubMed ID: 32695143
Summary:
Most eukaryotic genes express multiple transcripts and proteins, and a sophisticated gene expression strategy plays a crucial role in ensuring the cell-specificity of genetic information and the correctness of phenotypes. The Drosophila melanogaster gene shaggy encodes several isoforms of the conserved glycogen synthase kinase 3 (GSK3), which is vitally important for multiple biological processes. To characterize the phenotypic effects of differential shaggy expression, this study explored how the multidirectional modulation of the expression of the main GSK3 isoform, Shaggy-PB, in different tissues and cells affects lifespan. To this end, lines with transgenic constructs were used that encode mutant variants of the protein. The effect of shaggy misexpression on lifespan depended on the direction of the presumed change in GSK3 activity and the type of tissue/cell. The modulation of GSK3 activity in motor and dopaminergic neurons improved female lifespan but caused seemingly negative changes in the structural (mitochondrial depletion; neuronal loss) and functional (perturbed locomotion) properties of the nervous system, indicating the importance of analyzing the relationship between lifespan and healthspan in invertebrate models. These findings provide new insights into the molecular and cellular bases of lifespan extension, demonstrating that the fine-tuning of transcript-specific shaggy expression in individual groups of neurons is sufficient to provide a sex-specific increase in survival and slow aging.
Korona, D., Nightingale, D., Fabre, B., Nelson, M., Fischer, B., Johnson, G., Lees, J., Hubbard, S., Lilley, K. and Russell, S. (2020). Characterisation of protein isoforms encoded by the Drosophila Glycogen Synthase Kinase 3 gene shaggy. PLoS One 15(8): e0236679. PubMed ID: 32760087
Summary:
The Drosophila shaggy gene (sgg, GSK-3) encodes multiple protein isoforms with serine/threonine kinase activity and is a key player in diverse developmental signalling pathways. Currently it is unclear whether different Sgg proteoforms are similarly involved in signalling or if different proteoforms have distinct functions. This study used CRISPR/Cas9 genome engineering to tag eight different Sgg proteoform classes and determined their localization during embryonic development. Proteomic analysis was performed of the two major proteoform classes and mutant lines for both of these were generated for transcriptomic and phenotypic analysis. Distinct tissue-specific localization patterns were uncovered for all of the tagged proteoforms examined, most of which have not previously been characterised directly at the protein level, including one proteoform initiating with a non-standard codon. Collectively, this suggests complex developmentally regulated splicing of the sgg primary transcript. Further, affinity purification followed by mass spectrometric analyses indicate a different repertoire of interacting proteins for the two major proteoforms examined, one with ubiquitous expression (Sgg-PB) and one with nervous system specific expression (Sgg-PA). Specific mutation of these proteoforms shows that Sgg-PB performs the well characterised maternal and zygotic segmentations functions of the sgg locus, while Sgg-PA mutants show adult lifespan and locomotor defects consistent with its nervous system localisation. These findings provide new insights into the role of GSK-3 proteoforms and intriguing links with the GSK-3α and GSK-3β proteins encoded by independent vertebrate genes. This analysis suggests that different proteoforms generated by alternative splicing are likely to perform distinct functions.
Choi, H. J., Joo Cha, S., Do, H. A., Kim, H. J., Lee, J. W. and Kim, K. (2020). SCF-Slimb is critical for Glycogen synthase kinase-3beta-mediated suppression of TAF15-induced neurotoxicity in Drosophila. J Neurochem. PubMed ID: 32915460
Summary:
Amyotrophic lateral sclerosis (ALS) is a severe neurodegenerative disorder characterized pathologically by motor neuron degeneration and associated with aggregation of RNA-binding proteins. TATA-binding protein-associated factor 15 (TAF15) accumulates as cytoplasmic aggregates in neuronal cells, and clearance of these aggregates is considered a potential therapeutic strategy for ALS. However, the exact pathogenic mechanism of TAF15-induced neurotoxicity remains to be elucidated. Glycogen synthase kinase-3 (GSK-3) plays a critical role in the protection of ALS pathology. In this study, a transgenic fly model over-expressing human TAF15 was used to study the protective effects of Shaggy/GSK3β on TAF15-induced neuronal toxicity in Drosophila brain. Transgenic flies were examined for locomotor activity and lithium treatment. The expression level and solubility of TAF15 were assessed with western blotting, whereas immunohistochemistry was used to assess TAF15 aggregation in Drosophila brain. This study has revealed that Shaggy/GSK3β was abnormally activated in neurons of TAF15-expressing flies and its inhibition can suppress the defective phenotypes, thereby preventing retinal degeneration and locomotive activity caused by TAF15. It was also found that Shaggy/GSK3β inhibition in neuronal cells leads to a reduction in TAF15 levels. Indeed, the F-box proteins Slimb and archipelago genetically interact with TAF15 and control TAF15 protein level in Drosophila. Importantly, SCF(slimb) is a critical regulator for Shaggy/GSK3β-mediated suppression of TAF15-induced toxicity in Drosophila. The present study has provided an in vivo evidence supporting the molecular mechanism of GSK3β inhibition for protection against TAF15-linked proteinopathies.
Zhang, C., Seong, K. M., Sun, W., Mittapalli, O., Qiu, B., Clark, J. M. and Pittendrigh, B. R. (2021). The insulin signaling pathway in Drosophila melanogaster: A nexus revealing an "Achilles' heel" in DDT resistance. Pestic Biochem Physiol 171: 104727. PubMed ID: 33357549

Insecticide resistance is an ongoing challenge in agriculture and disease vector control. This study demonstrates a novel strategy to attenuate resistance. This study used genomics tools to target fundamental energy-associated pathways and identified a potential "Achilles' heel" for resistance, a resistance-associated protein that, upon inhibition, results in a substantial loss in the resistance phenotype. Specifically, the gene expression profiles and structural variations of the insulin/insulin-like growth factor signaling (IIS) pathway genes were compared in DDT-susceptible (91-C) and -resistant (91-R) Drosophila melanogaster (Drosophila) strains. A total of eight and seven IIS transcripts were up- and down-regulated, respectively, in 91-R compared to 91-C. A total of 114 nonsynonymous mutations were observed between 91-C and 91-R, of which 51.8% were fixed. Among the differentially expressed transcripts, phosphoenolpyruvate carboxykinase (PEPCK), down-regulated in 91-R, encoded the greatest number of amino acid changes, prompting the performance of PEPCK inhibitor-pesticide exposure bioassays. The inhibitor of PEPCK, hydrazine sulfate, resulted in a 161- to 218-fold decrease in the DDT resistance phenotype (91-R) and more than a 4- to 5-fold increase in susceptibility in 91-C. A second target protein, Glycogen synthase kinase 3β (GSK3β-PO), had one amino acid difference between 91-C and 91-R, and the corresponding transcript was also down-regulated in 91-R. A GSK3β-PO inhibitor, lithium chloride, likewise reduced the resistance but to a lesser extent than did hydrazine sulfate for PEPCK. This study has demonstrated the potential role of IIS genes in DDT resistance and the potential discovery of an "Achilles' heel" against pesticide resistance in this pathway (Zhang, 2021).

Pragati, S. S. (2021). Shaggy functions downstream of dMyc and their concurrent downregulation confers additive rescue against tau toxicity in Drosophila. Biofactors. PubMed ID: 33651466
Summary:
Neurodegenerative tauopathies such as Alzheimer's and Parkinson's diseases are characterized by hyperphosphorylation of tau protein and their subsequent aggregation in the forms of paired helical filaments and/or neurofibrillary tangles in specific areas of the brain. Despite several attempts, it remains a challenge to develop reliable biomarkers or effective drugs against tauopathies. It is increasingly evident now that due to the involvement of multiple cellular cascades affected by the pathogenic tau molecules, a single genetic modifier or a molecule is unlikely to be efficient enough to provide an inclusive rescue. Hence, multitargets based combinatorial approach(s) have been suggested to provide an efficient rescue against tauopathies. It has been reported that targeted downregulation of dmyc (a Drosophila homolog of human cmyc proto-oncogene) restricts tau etiology by limiting tau hyperphosphorylation and heterochromatin loss. Although, dmyc generates a significant rescue; however, it is not proficient enough to provide a complete alleviation against tauopathies. This study reports that tissue-specific concurrent downregulation of dmyc and gsk3β conveys a near-complete rescue against tau toxicity in Drosophila. It is noted that combinatorial downregulation of dmyc and gsk3β reduces tau hyperphosphorylation, restricts the formation of neurofibrillary tangles, and restores heterochromatin loss to the physiological level. Subsequent investigations revealed that dmyc regulates gsk3β via protein phosphatase 2A (dPP2A) in a dose-dependent manner to regulate tau pathogenesis. It is proposed that dmyc and gsk3β candidates can be utilized in a synergistic manner for the development of an efficient combinatorial therapeutic approach against the devastating human

BIOLOGICAL OVERVIEW

Phosphorylation is the main mechanism by which the cell transfers information from protein to protein along a signal transduction cascade. In the phosphorylation process, a phosphate residue is attached by means of an enzyme termed a kinase, to an amino acid residue of a downstream protein. Zeste-white 3, also known as Shaggy, is one of crucial kinases in the cell, and a critical one at that. Because for a long time, the Wingless receptor had not been identified, much research has focused on Shaggy, since Shaggy tranduces the wingless signal in the cell. Thus it is important to understand the place of Shaggy in the wingless signal transduction cascade.

What is the target of Shaggy phosphorylation? Wingless, through its receptor, inactivates shaggy. Genetic epistasis tests place shaggy upstream of armadillo (Peifer, 1994a and Sigfried, 1994). Mutations in shaggy mimic effects of the Wingless signal, resulting in an accumulation of high levels of cytoplasmic Arm in all cells (Peifer, 1994a). Together Shaggy/Zeste-white 3, and two other proteins, Axin and APC are termed the "ß-catenin destruction complex," which in Drosophila is responsible for degrading Armadillo, the ß-catenin homolog, thus releasing Pangolin, which subsequently enters the nucleus to activate Wingless target genes,

ARM is an integral part of the adherens junction as is ARM's closest vertebrate homolog, beta-catenin. The adherens junction is an adhesive contact point between cells, maintained by proteins that have connections across the membrane to the inside of the cell. Phosphorylation of ARM stabilizes its cytoplasmic form, where it may interact with other signal transduction molecules to pass the wingless signal into the nucleus (Peifer, 1994b). dishevelled and porcupine act upstream of shaggy. In porcupine mutants, Wingless appears confined to wingless expressing cells (Siegfried, 1994). Thus porcupine may be involved in Wingless secretion. dishevelled mutation adds no greater phenotypic effect to shaggy mutants, suggesting that dishevelled is upstream of shaggy, perhaps passing the signal from the Wingless receptor to the Shaggy kinase (Siegfried, 1994). Thus Shaggy is between the Dishevelled and Armadillo in the signal transduction pathway, receiving signals from Dishevelled and modifying Armadillo's association with junctions and its signaling process to the nucleus.

A model is presented for the role of Axin in Wnt signal transduction. In an unstimulated cell, GSK-3beta is active and phosphorylates Axin, which in turn, recruits beta-catenin into the Axin/GSK-3beta complex. By virtue of its proximity to GSK-3beta, beta-catenin is then phosphorylated. Phosphorylated beta-catenin is then targeted for degradation. Upon transduction of the Wnt signal through the Frizzled (Fz) receptors to Dishevelled, GSK-3beta kinase activity is inhibited so that PP2A (see Drosophila Twins) dephosphorylates Axin. Unphosphorylated Axin, in turn, no longer recruits beta-catenin to the complex. Failure of beta-catenin to associate with the Axin/GSK-3beta complex prevents its phosphorylation by GSK-3beta so that it can accumulate to high levels in the cytoplasm and nucleus and activate transcription in concert with the Tcf/Lef-1 family of transcription factors (Pangolin in Drosophila). GSK-3beta also phosphorylates APC, which may facilitate beta-catenin recruitment into the complex; however, this event has not been shown to be regulated by Wnt signaling (Willert, 1999).

Shaggy also appears to fill a 'neurogenic' role. At the site where neural precursors develop, achaete and scute are initially expressed in a group of equivalent cells (the proneural cluster). shaggy appears to act downstream of Notch for transduction of an inhibitory signal to adjacent cells. This second role of shaggy, now virtually ignored because of the current preoccupation with the function of Suppressor of hairless, may be due to modulation of some component of the Notch pathway (Simpson, 1993).

Work from Xenopus has clarified the interaction between Xgsk-3 (Shaggy in Drosophila) and ß-catenin (Armadillo in Drosophila). Xgsk-3 phosphorylates a ß-catenin serine/threonine rich site, which is conserved in the Drosophila protein Armadillo and in plakoglobin (a member of the same protein family). ß-catenin mutants lacking this site are more active in inducing an ectopic axis in Xenopus embryos and are more stable than wild-type ß-catenin in the presence of Xgsk-3 activity, supporting the hypothesis that Xgsk-3 is a negative regulator of ß-catenin, acting through the amino-terminal phosphorylation site. Xgsk-3 functions to destabilize ß-catenin and thus decrease the amount of ß-catenin available for signaling. The levels of endogenous ß-catenin in the nucleus increase in the presence of the dominant-negative Xgsk-3 mutant, suggesting that a role of Xgsk-3 is to regulate the steady-state levels of ß-catenin within specific subcellular compartments (Yost, 1996).

Work in the mouse suggests that protein kinase C (see Drosophila Protein kinase C) functions upstream of Shaggy. In mouse 10T1/2 fibroblasts, the activity of glycogen synthase kinase-3 (GSK-3), the murine homolog of Zw3/Sgg, is inactivated by Wingless. This occurs through a signaling pathway that is distinct from the insulin-mediated regulation of GSK-3, because Wg signaling to GSK-3 is insensitive to wortmannin. Wg-induced inactivation of GSK-3 is sensitive to both the protein kinase C (PKC) inhibitor Ro31-8220 and prolonged pre-treatment of 10T1/2 fibroblasts with phorbol ester. These findings provide the first biochemical evidence in support of the genetically defined pathway from Wg to Zw3/Sgg, and suggest a previously uncharacterized role for a PKC upstream of GSK-3/Zw3 during Wnt/Wg signal transduction (Cook, 1996).

The proposal that the Wingless signal is mediated by repression of Shaggy kinase activity was tested by overexpressing zeste white 3 in a tissue-specific fashion using the UAS/GAL4 binary expression system. Wild-type zw3 cDNA was placed under transcriptional control of the yeast GAL4 upstream activating sequence (UAS). UAS-zw3 flies were mated to flies that express the yeast transcriptional activator GAL4 in either a cell- or tissue-specific fashion to drive chronic expression of zw3. Elevated levels of zeste white 3 in the ectoderm and mesoderm result in phenotypes that resemble a loss of wingless. Overexpression of zeste white 3 in the mesoderm disrupts several Wingless-dependent processes, including the specification of a unique cell type in the larval midgut (the copper cell), the formation of the second midgut constriction, and the expression of Wingless target genes Ultrabithorax and decapentaplegic in the mesoderm, and labial in the endoderm. Interstitial cells normally found interspersed with the copper cells are still present. This loss of copper cells is similar to the phenotypes observed due to a loss of labial expression or wg expression, both required for the specification of the copper cells. The second midgut constriction is dependent on Wg signaling; in wg, dishevelled, or armadillo mutant embryos, this constriction does not form. Interestingly, in zw3 mutant embryos the second midgut constriction does form, but it is abnomal, appearing to have multiple folds. Zeste white 3 regulates the stability of Armadillo, which is essential for transducing the Wingless signal to the nucleus. zeste white 3 overexpression blocks Wingless signaling through the modulation of Armadillo since expression of a constitutively active form of Armadillo, which is independent of Zeste white 3 regulation, is epistatic to overexpression of zeste white 3 (Seitz, 1998).

Drosophila insulin and target of rapamycin (TOR) pathways regulate GSK3 beta activity to control Myc stability and determine Myc expression in vivo

Genetic studies in Drosophila reveal an important role for Myc in controlling growth. Similar studies have also shown how components of the insulin and target of rapamycin (TOR) pathways are key regulators of growth. Despite a few suggestions that Myc transcriptional activity lies downstream of these pathways, a molecular mechanism linking these signaling pathways to Myc has not been clearly described. Using biochemical and genetic approaches this study tried to identify novel mechanisms that control Myc activity upon activation of insulin and TOR signaling pathways. Biochemical studies show that insulin induces Myc protein accumulation in Drosophila S2 cells, which correlates with a decrease in the activity of glycogen synthase kinase 3-β (GSK3β) a kinase that is responsible for Myc protein degradation. Induction of Myc by insulin is inhibited by the presence of the TOR inhibitor rapamycin, suggesting that insulin-induced Myc protein accumulation depends on the activation of TOR complex 1. Treatment with amino acids that directly activate the TOR pathway results in Myc protein accumulation, which also depends on the ability of S6K kinase to inhibit GSK3β activity. Myc upregulation by insulin and TOR pathways is a mechanism conserved in cells from the wing imaginal disc, where expression of Dp110 and Rheb also induces Myc protein accumulation, while inhibition of insulin and TOR pathways result in the opposite effect. Functional analysis, aimed at quantifying the relative contribution of Myc to ommatidial growth downstream of insulin and TOR pathways, revealed that Myc activity is necessary to sustain the proliferation of cells from the ommatidia upon Dp110 expression, while its contribution downstream of TOR is significant to control the size of the ommatidia. This study presents novel evidence that Myc activity acts downstream of insulin and TOR pathways to control growth in Drosophila. At the biochemical level it was found that both these pathways converge at GSK3β to control Myc protein stability, while genetic analysis shows that insulin and TOR pathways have different requirements for Myc activity during development of the eye, suggesting that Myc might be differentially induced by these pathways during growth or proliferation of cells that make up the ommatidia (Parisi, 2011).

Previous studies in vertebrates have indicated a critical function for Myc downstream of growth factor signaling including insulin-like growth factor, insulin and TOR pathways. In Drosophila, despite a few notes that Myc transcriptional activity acts downstream of insulin and TOR pathways, no clear molecular mechanisms linking these pathways to Myc have been elucidated yet (Parisi, 2011).

It has been demonstrated that inhibition of GSK3β prevents Myc degradation by the proteasome pathway. This study further unravels the pathways that control Myc protein stability and shows that signaling by insulin and TOR induce Myc protein accumulation by regulating GSK3β activity in S2 cells. GSK3β is a constitutively active kinase that is regulated by multiple signals and controls numerous cellular processes. With the biochemical data it is proposed that GSK3β acts as a common point where insulin and TOR signaling converge to regulate Myc protein stability (see Model showing the proposed relationship between Myc and the insulin and TOR signaling pathways). In particular, activation of insulin signaling was shown to induce activation of Akt, an event that is accompanied by GSK3β phosphorylation on Ser 9 that causes its inactivation and Myc protein to stabilize. Interestingly, insulin-induced Myc protein accumulation, when GSK3β activity was blocked by the presence of LiCl or by expression of GSK3β-KD, was similar to that obtained with insulin alone. Since it was shown that activation of insulin signaling leads to GSK3β inhibition and to an increase in Myc protein, if insulin and GSK3β signaling were acting independently, it would be expected that activation of insulin signaling concomitantly with the inhibition of GSK3β activity would result in a higher level of Myc than that obtained with insulin or LiCl alone. The results instead showed a similar level of Myc protein accumulation with insulin in the presence of GSK3β inhibitors as compared to insulin alone, supporting the hypothesis that GSK3β and insulin signaling, at least in the current experimental condition, depend on each other in the mechanism that regulates Myc protein stability (Parisi, 2011).

In a similar biochemical approach, the effect of AAs was analyzed on Myc protein stability and how TOR signaling is linked to mechanisms that inactivate GSK3β to stabilize Myc protein in S2 cells. In these experiments it was possible to demonstrate that treatment with amino acids (AAs) increased Myc protein stability, and it was also shown that treatment with rapamycin, an inhibitor of TORC1, reduced insulin-induced Myc upregulation. The reduction of Myc protein accumulation by rapamycin was blocked by inhibition of the proteasome pathway, linking TOR signaling to the pathway that controls Myc protein stability. TORC1 is a central node for the regulation of anabolic and catabolic processes and contains the central enzyme Rheb-GTPase, which responds to amino acids by activating TOR kinase to induce phosphorylation of p70-S6K and 4E-BP1. Analysis of the molecular mechanisms that act downstream of TOR to regulate Myc stability shows that AA treatment induces p70-S6K to phosphorylate GSK3β on Ser 9, an event that results in its inactivation and accumulation of Myc protein (Parisi, 2011).

Reducing GSK3β activity with LiCl, in medium lacking AAs, resulted in a slight increase in Myc protein levels. Adding back AAs lead to a substantial increase in Myc protein levels, which did not further increase when AAs where added to cells in the presence of the GSK3β inhibitor LiCl. These events were accompanied by phosphorylation of S6K on Thr 398, which correlated with phosphorylation of GSK3β on Ser 9. From these experiments it is concluded that TOR signaling also converges to inhibit GSK3β activity to regulate Myc protein stability. However, it has to be pointed out that since AAs alone increased Myc protein levels to a higher extent than that observed with LiCl alone, the experiments also suggest that Myc protein stability by TOR signaling is not solely directed through the inhibition of GSK3β activity, but other events and/or pathways contribute to Myc regulation. In conclusion, the biochemical experiments demonstrate that GSK3β acts downstream of insulin and TOR pathways to control Myc stability, however it cannot be excluded that other pathways may control Myc protein stability upon insulin and amino acids stimulation in S2 cells (Parisi, 2011).

Reduction of insulin and TOR signaling in vivo reduces cell size and proliferation, and clones mutant for chico, the Drosophila orthologue of IRS1-4, or for components of TOR signaling, are smaller due a reduction in size and the number of cells. The experiments showed that reducing insulin signaling by expression of PTEN or using TORTED, a dominant negative form of TOR, decreased Myc protein levels in clones of epithelial cells of the wing imaginal discs, while the opposite was true when these signals were activated using Dp110 or RhebAV4 . Those experiments suggested that the mechanism of regulation of Myc protein by insulin and TOR pathways was conserved also in vivo in epithelial cells of the larval imaginal discs (Parisi, 2011).

During these experiments it was also noted that Myc protein was induced in the cells surrounding and bordering the clones (non-autonomously), particularly when clones where positioned along the dorsal-ventral axis of the wing disc. This upregulation of Myc protein was not restricted to components of the insulin signaling pathway since it was also observed in cells surrounding the clones mutant for components of the Hippo pathway or for the tumor suppressor lethal giant larvae (lgl), which upregulates Myc protein cell-autonomously. It is suspected that this non-autonomous regulation of Myc may be induced by a novel mechanism that controls proliferation of cells when 'growth' is unbalanced. It can be speculated that clones with different growth rates, caused by different Myc levels, might secrete factors to induce Myc expression in neighboring cells. As a consequence, these Myc-expressing cells will speed up their growth rate in an attempt to maintain proliferation and tissue homeostasis. Further analysis is required to identify the mechanisms responsible for this effect (Parisi, 2011).

In order to distinguish if Myc activity was required downstream of insulin and TOR signaling to induce growth, a genetic analysis was performed. The ability to induce growth and proliferation was measured in the eye by measuring the size and number of the ommatidia from animals expressing members of the insulin and TOR pathways in different dm genetic background (dm+, dmP0 and dm4). The data showed that Dp110 increased the size and number of the ommatidia, however only the alteration in the total number was dependent on dm levels. These data suggest that Myc is required downstream of insulin pathway to achieve the proper number of ommatidia. However, when insulin signaling was reduced by PTEN, a significant decrease in the size of ommatidia was seen and it was dependent on dm expression levels, suggesting that Myc activity is limiting for ommatidial size and number. Activation of TOR signaling induces growth, and the genetic analysis showed that Myc significantly contributes to the size of the ommatidial cells thus suggesting that Myc acts downstream of TOR pathway to control growth (Parisi, 2011).

Recent genomic analysis showed a strong correlation between the targets of Myc and those of the TOR pathway, implying that they may share common targets. In support of this observation, mosaic analysis with a repressible cell marker (MARCM) experiments in the developing wing disc showed that overexpression of Myc partially rescues the growth disadvantage of clones mutant for the hypomorphic Rheb7A1 allele, further supporting the idea that Myc acts downstream of TOR to activate targets that control growth in these clones (Parisi, 2011).

The genetic interaction revealed a stronger dependence on Myc expression when Rheb was used as opposed to S6K. A possible explanation for this difference could lie in the fact that S6K is not capable of auto-activation of its kinase domain unless stimulated by TOR kinase. TOR activity is dependent on its upstream activator Rheb; consequently the enzymatic activity of the Rheb/GTPase is the limiting factor that influences S6K phosphorylation and therefore capable of maximizing its activity (Parisi, 2011).

Interestingly, these experiments also showed that activation of TOR signaling has a negative effect on the number of ommatidia, and this correlates with the ability of RhebAV4 to induce cell death during the development of the eye imaginal disc. Rheb-induced cell death was rescued in a dmP0 mutant background, which leading to the speculation that 'excessive' protein synthesis, triggered by overexpression of TOR signaling, could elicit a Myc-dependent stress response, which induces apoptosis. Alternatively, high protein synthesis could result in an enrichment of misfolded proteins that may result in a stress response and induces cell death. Further analysis is required to delineate the mechanisms underlying this process (Parisi, 2011).

This analyses provide novel genetic and biochemical evidences supporting a role for Myc in the integration of the insulin and TOR pathway during the control of growth, and highlights the role of GSK3β in this signaling. It was found that insulin signaling inactivates GSK3β to control Myc protein stability, and a similar biochemical regulation is also shared by activation of the TOR pathways. In support of this data, a recent genomic analysis in whole larvae showed a strong correlation between the targets of Myc and those of the TOR pathway; however, less overlap was found between the targets of Myc and those of PI3K signaling (Parisi, 2011).

Statistical analysis applied to the genetic interaction experiments revealed that, in the Drosophila eye, proliferation induced by activation of the insulin pathway is sensitive to variations in Myc levels, while a significant interaction was seen mostly when TOR increased cell size. The data therefore suggests that there is a correlation between Myc and the InR signaling and it is expected that the InR pathway also shares some transcriptional targets with Myc. Indeed, an overlap was found between the targets induced by insulin and Myc in Drosophila S2 cells and these targets have also been reported in transcriptome analyses in the fat body upon nutritional stress, suggesting that Myc acts downstream of InR/PI3K and TOR signaling and that this interaction might be specific to some tissues or in a particular metabolic state of the cell (Parisi, 2011).

Nebula/DSCR1 upregulation delays neurodegeneration and protects against APP-induced axonal transport defects by restoring calcineurin and GSK-3beta signaling

Post-mortem brains from Down syndrome (DS) and Alzheimer's disease (AD) patients show an upregulation of the Down syndrome critical region 1 protein (DSCR1), but its contribution to AD is not known. To gain insights into the role of DSCR1 in AD, this study explored the functional interaction between DSCR1 and the amyloid precursor protein (APP), which is known to cause AD when duplicated or upregulated in DS. The Drosophila homolog of DSCR1, Nebula, was found to delay neurodegeneration and ameliorates axonal transport defects caused by APP overexpression. Live-imaging reveals that Nebula facilitates the transport of synaptic proteins and mitochondria affected by APP upregulation. Furthermore, Nebula upregulation was shown to protect against axonal transport defects by restoring calcineurin and GSK-3beta signaling altered by APP overexpression, thereby preserving cargo-motor interactions. As impaired transport of essential organelles caused by APP perturbation is thought to be an underlying cause of synaptic failure and neurodegeneration in AD, these findings imply that correcting calcineurin and GSK-3beta signaling can prevent APP-induced pathologies. The data further suggest that upregulation of Nebula/DSCR1 is neuroprotective in the presence of APP upregulation and provides evidence for calcineurin inhibition as a novel target for therapeutic intervention in preventing axonal transport impairments associated with AD (Shaw, 2013).

Although upregulation of APP had been shown to negatively influence axonal transport in mouse and fly models, mechanisms by which APP upregulation induces transport defects are poorly understood. Several hypotheses have been proposed, including titration of motor/adaptor by APP, impairments in mitochondrial bioenergetics, altered microtubule tracks, or aberrant activation of signaling pathways. The motor/adaptor titration theory suggests that excessive APP-cargos titrates the available motors away from other organelles, thus resulting in defective transport of pre-synaptic vesicles. The finding that Nebula co-upregulation enhanced the movement and delivery of both synaptotagmin and APP to the synaptic terminal argues against this hypothesis. In addition, earlier findings suggest that Nebula upregulation alone impaired mitochondrial function and elevated ROS level, thus implying that Nebula is not likely to rescue APP-dependent phenotypes by selectively restoring mitochondrial bioenergetics. Furthermore, consistent with a recent report showing normal microtubule integrity in flies overexpressing either APP-YFP or activated GSK-3βM (Weaver, 2013), the data revealed normal gross microtubule structure in flies with APP overexpression. Together, these results suggest that changes in gross microtubule structure and stability is not a likely cause of APP-induced transport defects (Shaw, 2013).

Instead, the current results support the idea that Nebula facilitates axonal transport defects by correcting APP-mediated changes in phosphatase and kinase signaling pathways. First, APP upregulation was found to elevate intracellular calcium level and calcineurin activity, and restoring calcineurin activity to normal suppresses the synaptotagmin aggregate accumulation in axons. The observed increase in calcium and calcineurin activity is consistent with reports of calcium dyshomeostasis and elevated calcineurin phosphatase activity found in AD brains, as well as reports demonstrating elevated neuronal calcium level due to APP overexpression and increased calcineurin activation in Tg2576 transgenic mice carrying the APPswe mutant allele. Second, APP upregulation resulted in calcineurin dependent dephosphorylation of GSK-3β at Ser9 site, a process thought to activate GSK-3β kinase. APP upregulation also triggered calcineurin-independent phosphorylation at Tyr216 site, which has been shown to enhance GSK-3β activity. The kinase(s) that phosphorylates APP at Tyr216 is currently not well understood, it will be important to study how APP leads to Tyr216 phosphorylation in the future. Based on the current results, it is envisioned that APP overexpression ultimately leads to excessive calcineurin and GSK-3β activity, whereas nebula overexpression inhibits calcineurin to prevent activation of GSK-3β. The findings that nebula co-overexpression prevents GSK-3β activation and enhances the transport of APP-YFP vesicles are consistent with a recent report by Weaver (2013), in which it was found decreasing GSK-3β in fly increases the speed of APP-YFP movement. Furthermore, consistent with the current result that APP upregulation triggers GSK-3β enhancement and severe axonal transport defect, Weaver did not detect changes in GFP-synaptotagmin movement in the absence of APP upregulation (Shaw, 2013).

Active GSK-3β has been shown to influence the transport of mitochondria and synaptic proteins including APP, although the exact mechanism may differ between different cargos and motors. One mechanism proposed for GSK-3β-mediated regulation of axonal transport is through phosphorylation of KLC1, thereby disrupting axonal transport by decreasing the association of the anterograde molecular motor with its cargos. Accordingly, this study found that APP reduces KLC-synaptotagmin interaction while Nebula upregulation preserves it. Synaptotagmin transport in both the anterograde and retrograde directions are affected, consistent with previous reports showing that altering either the anterograde kinesin or retrograde dynein is sufficient affected transport in both directions. The results also support work suggesting that synaptotagmin can be transported by the kinesin 1 motor complex in addition to the kinesin 3/imac motor. As kinesin 1 is known to mediate the movement of both APP and mitochondria and phosphorylation of KLC had been shown to inhibit mitochondrial transport, detachment of cargo-motor caused by GSK-3β mediated phosphorylation of KLC may lead to general axonal transport problems as reported in this study. However, GSK-3β activation may also perturb general axonal transport by influencing motor activity or binding of motors to the microtubule tract. Interestingly, increased levels of active GSK-3β and phosphorylated KLC and dynein intermediate chain (DIC), a component of the dynein retrograde complex, have been observed in the frontal complex of AD patients. Genetic variability for KLC1 is thought to be a risk factor for early-onset of Alzheimer's disease. There is also increasing evidence implicating GSK-3β in regulating transport by modulating kinesin activity and exacerbating neurodegeneration in AD through tau hyperphosphorylation. It will be interesting to investigate if Nebula also modulates these processes in the future (Shaw, 2013).

SAlthough calcineurin had been shown to regulate many important cellular pathways, the link between altered calcineurin and axonal transport, especially in the context of AD, had not been established before. This study shows that calcineurin can regulate axonal transport through both GSK-3β independent and dependent pathways. This is supported by observation that the severity of the aggregate phenotype was worse for flies expressing APP and active calcineurin than it was for flies expressing APP and active GSK-3β. These findings point to a role for calcineurin in influencing axonal transport directly, perhaps through dephosphorylation of motor or adaptor proteins. The data also indicate that calcineurin in part modulates axonal transport through dephosphorylation of GSK-3β as discussed above; however, upregulation of APP is necessary for the induction of severe axonal transport problems, mainly by causing additional enhancement of GSK-3β signaling. GSK3 inhibition is widely discussed as a potential therapeutic intervention for AD; results suggest that perhaps calcineurin is a more effective target for delaying degeneration by preserving axonal transport (Shaw, 2013).

DSCR1 and APP are both located on chromosome 21 and upregulated in DS. Overexpression of DSCR1 alone had been contradictorily implicated in both conferring resistance to oxidative stress and in promoting apoptosis. Upregulation of Nebula/DSCR1 had also been shown to negatively impact learning and memory in fly and mouse models through altered calcineurin pathways. How could upregulation of DSCR1 be beneficial? It is proposed that DSCR1 upregulation in the presence of APP upregulation compensates for the altered calcineurin and GSK-3β signaling, shifting the delicate balance of kinase/phosphatase signaling pathways close to normal, therefore preserving axonal transport and delaying neurodegeneration. It is also proposed that axonal transport defects and synapse dysfunction caused by APP upregulation in the Drosophila model system occur prior to accumulation of amyloid plaques and severe neurodegeneration, similar to that described for a mouse model (Shaw, 2013).

DS is characterized by the presence of AD neuropathologies early in life, but most DS individuals do not exhibit signs of dementia until decades later, indicating that there is a delayed progression of cognitive declin. The upregulation of DSCR1 may in fact activate compensatory cell signaling mechanisms that provide protection against APP-mediated oxidative stress, aberrant calcium, and altered calcineurin and GSK3-β activity (Shaw, 2013).


GENE STRUCTURE

shaggy has multiple transcripts and is alternatively spliced. Of ten developmentally regulated transcripts, two seem to be responsible for early sgg activity. There are five different proteins generated by alternative splicing (Bourouis, 1990 and Ruel, 1993).

Length of genomic DNA - 40 kb

Exons - There are nine, whose translation products are incorporated in various combinations in the different protein transcripts.


PROTEIN STRUCTURE

Structural Domains

Shaggy has a catalytic domain found in serine/threonine specific protein kinases, linked to a region unusually rich in Gly, Ala and Ser (Bourouis, 1990).


shaggy: Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 5 April 2021 

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