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 links: Precomputed BLAST | Entrez Gene

Recent literature
Mannava, A. G. and Tolwinski, N. S. (2015). Membrane bound GSK-3 activates Wnt signaling through Disheveled and Arrow. PLoS One 10: e0121879. PubMed ID: 25848770
Summary:
Wnt ligands and their downstream pathway components coordinate many developmental and cellular processes. In adults, they regulate tissue homeostasis through regulation of stem cells. Mechanistically, signal transduction through this pathway is complicated by pathway components having both positive and negative roles in signal propagation. This study examined the positive role of GSK-3/Zw3 in promoting signal transduction at the plasma membrane. Targeting GSK-3 to the plasma membrane activates signaling in Drosophila embryos. This activation requires the presence of the co-receptor Arrow-LRP5/6 and the pathway activating protein Disheveled. These results provide genetic evidence for evolutionarily conserved, separable roles for GSK-3 at the membrane and in the cytosol, and are consistent with a model where the complex cycles from cytosol to membrane in order to promote signaling at the membrane and to prevent it in the cytosol.

Moncrieff, S., Moncan, M., Scialpi, F. and Ditzel, M. (2015). Regulation of Hedgehog ligand expression by the N-end rule ubiquitin-protein ligase Hyperplastic discs and the Drosophila GSK3β homologue, Shaggy. PLoS One 10: e0136760. PubMed ID: 26334301
Summary:

Hedgehog (Hh) morphogen signalling plays an essential role in tissue development and homeostasis. While much is known about the Hh signal transduction pathway, far less is known about the molecules that regulate the expression of the hedgehog (hh) ligand itself. This study revealed that Shaggy (Sgg), the Drosophila melanogaster orthologue of GSK3β, and the N-end Rule Ubiquitin-protein ligase Hyperplastic Discs (Hyd) act together to co-ordinate Hedgehog signalling through regulating hh ligand expression and Cubitus interruptus (Ci) expression. Increased hh and Ci expression within hyd mutant clones was effectively suppressed by sgg RNAi, placing sgg downstream of hyd. Functionally, sgg RNAi also rescued the adult hyd mutant head phenotype. Consistent with the genetic interactions, Hyd esd found to physically interact with Sgg and Ci. Taken together it iw proposed that Hyd and Sgg function to co-ordinate hh ligand and Ci expression, which in turn influences important developmental signalling pathways during imaginal disc development. These findings are important as tight temporal/spatial regulation of hh ligand expression underlies its important roles in animal development and tissue homeostasis. When deregulated, hh ligand family misexpression underlies numerous human diseases (e.g., colorectal, lung, pancreatic and haematological cancers) and developmental defects (e.g., cyclopia and polydactyly). In summary, these Drosophila-based findings highlight an apical role for Hyd and Sgg in initiating Hedgehog signalling, which could also be evolutionarily conserved in mammals.

Sieber, M. H., Thomsen, M. B. and Spradling, A. C. (2016). Electron transport chain remodeling by GSK3 during oogenesis connects nutrient state to reproduction. Cell 164: 420-432. PubMed ID: 26824655
Summary:
Reproduction is heavily influenced by nutrition and metabolic state. Many common reproductive disorders in humans are associated with diabetes and metabolic syndrome. This study characterized the metabolic mechanisms that support oogenesis and found that mitochondria in mature Drosophila oocytes enter a low-activity state of respiratory quiescence by remodeling the electron transport chain (ETC). This shift in mitochondrial function leads to extensive glycogen accumulation late in oogenesis and is required for the developmental competence of the oocyte. Decreased insulin signaling initiates ETC remodeling and mitochondrial respiratory quiescence through glycogen synthase kinase 3 (GSK3). Intriguingly, similar ETC remodeling and glycogen uptake was observed in maturing Xenopus oocytes, suggesting that these processes are evolutionarily conserved aspects of oocyte development. These studies reveal an important link between metabolism and oocyte maturation.

Castillo-Quan, J.I., Li, L., Kinghorn, K.J., Ivanov, D.K., Tain, L.S., Slack, C., Kerr, F., Nespital, T., Thornton, J., Hardy, J., Bjedov, I. and Partridge, L. (2016). Lithium promotes longevity through GSK3/NRF2-dependent hormesis. Cell Rep [Epub ahead of print]. PubMed ID: 27068460
Summary:
The quest to extend healthspan via pharmacological means is becoming increasingly urgent, both from a health and economic perspective. This study shows that lithium, a drug approved for human use, promotes longevity and healthspan. Lithium was shown to extend lifespan in female and male Drosophila, when administered throughout adulthood or only later in life. The life-extending mechanism involves the inhibition of glycogen synthase kinase-3 (GSK-3) and activation of the transcription factor nuclear factor erythroid 2-related factor (NRF-2). Combining genetic loss of the NRF-2 repressor Kelch-like ECH-associated protein 1 (Keap1) with lithium treatment revealed that high levels of NRF-2 activation confer stress resistance, while low levels additionally promote longevity. The discovery of GSK-3 as a therapeutic target for aging will likely lead to more effective treatments that can modulate mammalian aging and further improve health in later life.

Top, D., Harms, E., Syed, S., Adams, E. L. and Saez, L. (2016). GSK-3 and CK2 kinases converge on Timeless to regulate the master clock. Cell Rep [Epub ahead of print]. PubMed ID: 27346344
Summary:
The molecular clock relies on a delayed negative feedback loop of transcriptional regulation to generate oscillating gene expression. Although the principal components of the clock are present in all circadian neurons, different neuronal clusters have varying effects on rhythmic behavior, suggesting that the clocks they house are differently regulated. Combining biochemical and genetic techniques in Drosophila, this study identified a phosphorylation program native to the master pacemaker neurons that regulates the timing of nuclear accumulation of the Period/Timeless repressor complex. GSK-3/SGG binds and phosphorylates Period-bound Timeless, triggering a CK2-mediated phosphorylation cascade. Mutations that block the hierarchical phosphorylation of Timeless in vitro also delay nuclear accumulation in both tissue culture and in vivo and predictably change rhythmic behavior. This two-kinase phosphorylation cascade is anatomically restricted to the eight master pacemaker neurons, distinguishing the regulatory mechanism of the molecular clock within these neurons from the other clocks that cooperate to govern behavioral rhythmicity.
Iacobucci, G. J. and Gunawardena, S. (2017). Ethanol stimulates the in vivo axonal movement of neuropeptide dense core vesicles in Drosophila motor neurons. J Neurochem [Epub ahead of print]. PubMed ID: 28960313
Summary:
Proper neuronal function requires essential biological cargoes to be packaged within membranous vesicles and transported, intracellularly, through the extensive outgrowth of axonal and dendritic fibers. The precise spatiotemporal movement of these cargoes is vital for neuronal survival and, thus, is highly regulated. This study tested how the axonal movement of a neuropeptide containing dense core vesicle (DCV) responds to alcohol stressors. Ethanol was found to induce a strong anterograde bias in vesicle movement. Low doses of ethanol stimulate the anterograde movement of neuropeptide-DCV while high doses inhibit bidirectional movement. This process required the presence of functional kinesin-1 motors as reduction of kinesin prevented the ethanol-induced stimulation of the anterograde movement of neuropeptide-DCV. Furthermore, expression of inactive GSK-3beta also prevented ethanol-induced stimulation of neuropeptide-DCV movement, similar to pharmacological inhibition of GSK-3beta with lithium. Conversely, inhibition of PI3K/AKT signaling with wortmannin led to a partial prevention of ethanol-stimulated transport of neuropeptide-DCV. Taken together, it is concluded that GSK-3beta signaling mediates the stimulatory effects of ethanol. Therefore, this study provides new insight into the physiological response of the axonal movement of neuropeptide-DCV to exogenous stressors.

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).

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: 26 FEB 97 

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