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).

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).

date revised: 26 FEB 97 

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