BIOLOGICAL OVERVIEW

In Drosophila, Dishevelled is required in two distinct signaling pathways that share Frizzled receptors of similar structure, but have distinct intracellular signaling routes. Dishevelled phosphorylation is concurrent with Wingless pathway activation. Wingless serves to activate Dsh, which in turn inactivates Shaggy/Zeste white 3. In the absence of signaling, Armadillo is targeted for proteolytic degradation through phosphorylation by Shaggy. Arm accumulates in the cytoplasm and nucleus, where it activates Wg target genes by complexing with Pangolin/TCF transcription factors. Experiments suggest that upon Wnt signaling, part of the cytoplasmic pool of Dsh is phosphorylated and recruited to the membrane. Deletion analysis experiments have shown that the Dsh DEP domain is not required to induce Arm accumulation. The Dishevelled role in the tissue polarity pathway clearly indicates that, compared to its role in the Wingless pathway, different downstream components are involved. Rather, dominant genetic interactions and rescue experiments in the eye imaginal disc suggest that signaling is routed through a Rho/Rac and JNK/SAPK-like kinase cascade. Thus, Dsh is involved in two distinct Wnt signaling pathways, raising the question of how Dsh routes the information to the right effector cascade in each case (Boutros, 1999 and references).

dishevelled is an integral part of the wingless pathway. Wingless is secreted from anterior segmental compartments (for details, see wingless and engrailed), and is induced by Hedgehog, a protein made in posterior compartments. Wingless is required for the maintenance of engrailed synthesis, and for the accumulation of Armadillo protein (Klingensmith, 1994). Cells lacking dsh are unable to adopt the fates specified by Wingless.

Dishevelled is one element in a chain of interacting proteins that carry the wingless signal to sites within the cell, including the cell membrane and the nucleus. Phosphorylation is the most important biochemical mechanism for carrying signals between proteins. Dishevelled, a phosphoprotein, is synthesized uniformly throughout the embryo, but its level of phosphorylation varies with the level of wingless activity, (Yanagawa, 1995). Evidence that the serine threonine kinase shaggy/zeste white-3 is downstream of dishevelled suggests that it is not this kinase that is responsible for DSH phosphorylation, but that DSH phosphorylation is carried out by an as yet unidentified kinase (Yanagawa, 1995).

Phosphorylated DSH is correlated with high levels of cytoplasmic phosphorylated Armadillo. The function of this modification of ARM is not yet understood (Peifer, 1994), but certainly DSH and the wingless pathway are involved in cell surface integrity, acting through Armadillo on adherens junction stability (Klingensmith, 1994).

Most if not all biological organisms demonstrate an incredible (though imperfect) symmetry of structure and patterning. By symmetry is meant the positional repetition of a pattern on opposite sides of a dividing line or plane, distributed about a center or axis, resulting in geometric regularity. Symmetry is only one aspect of patterning. A second aspect, tissue polarity is exemplified by the pattern of epithelial hairs. For example, all the hairs of the wing surface point in the same direction. What is the biological basis of tissue symmetry and polarity? In Drosophila, planar cell polarity (PCP) signaling is mediated by the receptor Frizzled (Fz) and transduced by Dishevelled (Dsh). PCP signaling controls the polarity of epithelial cells within a plane orthogonal to their apical-basal axis. One manifestation of this cellular polarity is the oriented organization of trichomes (cell hairs). In wild-type flies, the cell hair arising from each cell's distal vertex contributes to a parallel and specifically oriented array. Mutations in dsh, as well as additional genes including frizzled (fz), prickle (pk),inturned (in), fuzzy (fy),multiple wing hairs (mwh), and others all disrupt the polarity of the trichomes. The resulting mutant phenotypes include swirls and distortions of the hair polarity pattern, and in some instances, more than one trichome per cell. A putative signal transduction pathway has been proposed, which serves to polarize cells, allowing them to distinguish one side of the cell from the other, and to propagate this information from cell to cell. In this pathway, Frizzled, a seven-transmembrane protein (without apparent primary sequence homology to the G-protein-coupled receptors) acts as a receptor, functioning upstream of Dsh. Dsh then antagonizes the activities of Fuzzy, Inturned (both novel transmembrane proteins), and Mwh; in turn, it is proposed that these proteins regulate the cytoskeletal apparatus responsible for control of PCP. Mutations in RhoA affect this process, and experiments with dominant-negative mutants have implicated Cdc42 and Rac1 as additional effectors. Pk is proposed to function either in transmission of the signal to adjacent cells, or in interpretation of the directionality of the signal. Thus far, no ligand for the PCP pathway has been identified. Although a tentative signal transduction pathway has been proposed, the mechanism by which asymmetry is established in the responding cells is not understood (Axelrod and references, 1998).

In Drosophila, planar cell polarity signaling is mediated by the receptor Frizzled (Fz) and transduced by Dishevelled (Dsh). Wingless (Wg) signaling also requires Dsh and may utilize DFz2 as a receptor. A heterologous system was used to examine the interaction of Dsh with Frizzled. mRNAs encoding Fz or Frizzled2 and a fusion of Dsh to green fluorescent protein (Dsh-GFP) were synthesized in vitro and injected into Xenopus embryos at the four-cell stage. Animal caps from stage 9 embryos, dissected to reveal the blastocoelar cells, were then examined by confocal microscopy. Dsh is recruited selectively to the membrane by Fz but not Frizzled2, and this recruitment depends on the DEP domain but not the PDZ domain in Dsh. When Fz is expressed simultaneously with Dsh-GFP, Dsh-GFP shows a qualitative redistribution to the membrane or cell cortex. Under these conditions, localization of Dsh-GFP to filopodia present on the blastocoelar (free) surfaces of the animal cap cells was also noticed. Staining with phalloidin (and Dsh-GFP) revealed that the filopodia contain filamentous actin. It is interesting to note that although the filopodia stain with Dsh-GFP, little or no Fz localizes there; at the cell cortex, the Fz and Dsh-GFP show imperfect colocalization. Fz staining is localized predominantly to the plasma membrane, and to a lesser extent to intracellular membranes (probably ER and/or Golgi) in these cells. This suggests that while Fz may induce localization of Dsh-GFP to the membrane and filopodia, it may do so by a mechanism other than direct binding. Frizzled2, the Wingless receptor, fails to induce membrane localization of Dsh, even in the presence of a functional Fz2 ligand (Axelrod, 1998).

Drosophila Dsh is a modular protein of unknown function that is well conserved in relation to its vertebrate homologs. Alignment of family members reveals three conserved domains. The first, a DIX domain, is similar to a domain in murine Axin (see Drosophila Axin), a recently described modulator of the Wnt1 pathway. The second contains a PDZ domain; PDZ domains recognize and bind short motifs at the carboxyl termini of proteins (but may bind other motifs as well). PDZ domains can also form dimers. The third domain, called DEP, is conserved among a set of proteins that have in common the ability to regulate various GTPases, including both heterotrimeric G proteins and Ras-like small GTPases. A mutation in the DEP domain impairs both membrane localization and the function of Dsh in PCP signaling, indicating that translocation is important for function. A single amino acid substitution in the DEP domain of Dsh is shown to confer a loss of function for PCP signaling, yet the mutant protein is functional for Wg signaling. This single amino acid substitution, coded for by the dsh¹ allele, allows for translocation to the membrane, but is thought to impair the ability of Dsh¹ to associate with its target at the membrane. This altered membrane interaction diminishes the ability of Dsh¹ to function in PCP signaling (Axelrod, 1998).

Further genetic and molecular analyses suggest that conserved domains in Dsh function differently during PCP and Wg signaling, and that divergent intracellular pathways are activated. For example, the individual domains Dsh(DIX) and Dsh deleted for the PDZ domain, are each dominant negative for Wg signaling but have no effect on PCP signaling. Overexpression of Zeste white3, or an activated Arm protein, both involved in Wingless signaling, also fail to produce any effect on PCP. It is proposed that Dsh has distinct roles in PCP and Wg signaling. The PCP signal may selectively result in focal Fz activation and asymmetric relocalization of Dsh to the membrane, where Dsh effects cytoskeletal reorganization to orient prehair initiation. This analysis suggests that Dsh has different interactions in PCP and Wg signaling and predicts an additional genetic behavior (Axelrod, 1998).

If Wg and planar polarity signaling utilize Dsh in a common fashion, then ectopic activation of one pathway should be able to cross-activate the other by promiscuously activating Dsh. In contrast, if each pathway utilizes Dsh in a distinct fashion, then ectopic activation might sequester Dsh in pathway-specific complexes, rendering it unavailable and therefore titrating the activity of the other pathway. These possibilities could best be tested under conditions in which Dsh is limiting. Overexpression of Fz causes a dominant gain-of-function PCP phenotype, and this phenotype is sensitive to the dose of dsh. Can Wg cross-activate Dsh activity for PCP signaling, or can it sequester Dsh? To investigate this question, it was first necessary to know if the Fz-overexpression phenotype would be either enhanced or suppressed by Wingless overexpression. Ectopic expression of Wg suppresses the Fz overexpression phenotype, suggesting that activation of Wg signaling may titrate the amount of Dsh available for PCP signaling. The reciprocal experiment was performed by asking if ectopic activation of the PCP pathway could interfere with Wg signaling. Because the ligand for PCP signaling is unknown, Fz was overexpressed during embryogenesis, and the cuticle phenotype analyzed. These embryos develop with lawns of denticles and are reminiscent of wg-mutant embryos, or those expressing dominant-negative Dsh constructs. The results suggest that titration can occur in this direction as well. The possibility cannot be ruled out that the titration observed in these experiments results from a promiscuous interaction between Wg and Fz, although this interaction may not occur in vivo. The observations are equally consistent with the possibility that under these conditions, activity of one pathway titrates the Dsh level available for the other (Axelrod, 1998).

Dishevelled appears to interact with the Notch pathway, and this interaction provides a direct link between Notch and wingless pathways. The dishevelled gene interacts antagonistically with Notch and its ligand Delta. A direct physical interaction between Dishevelled and the Notch carboxyl terminus, distal to the cdc10/ankyrin repeats, suggests a mechanism for this interaction. It is proposed that Dishevelled, in addition to transducing the Wingless signal, blocks Notch signaling directly, thus providing a molecular mechanism for the inhibitory cross talk observed between these pathways (Axelrod, 1996).

Frizzled family proteins have been described as receptors of Wnt signaling molecules. In Drosophila, the two known Frizzled proteins are associated with distinct developmental processes. Genesis of epithelial planar polarity requires Frizzled, whereas Dfz2 affects morphogenesis by wingless-mediated signaling. Dishevelled is required in both signaling pathways. Genetic and overexpression assays have been used to show that Dishevelled activates JNK cascades. In contrast to the action of wingless-pathway components, mutations in rhoA, hemipterous, basket, and jun as well as deficiencies removing the Rac1 and Rac2 genes show a strong dominant suppression of a Dishevelled overexpression phenotype in the compound eye. In an in vitro assay, expression of Dsh has been shown to induce phosphorylation of Jun, indicating that Dsh is a potent activator of the JNK pathway. Whereas the PDZ domain of Dsh, known to be required in the transduction of the wingless signal, is dispensable for signal-independent induction of Jun phosphorylation, the C-terminal DEP domain of Dsh is found to be essential. The planar polarity-specific dsh1 allele is found to be mutated in the DEP domain. These results indicate that different Wnt/Fz signals activate distinct intracellular pathways, and Dishevelled discriminates among them by distinct domain interactions (Boutros, 1998).

How can Fz/Dsh signaling be linked to small GTPase and JNK/MAPK pathways? Recent studies provided evidence that links G protein-coupled receptors, which share structural features with Fz proteins, to MAPK signaling through heterotrimeric G proteins and PI-3 kinases. It is intriguing to speculate that a subset of Fz proteins might signal through a similar pathway. It was also shown recently that XWnt5A and rFz2, in a heterologous assay, increase intracellular calcium via G proteins and phosphoinositol signaling. A mutation in the beta-subunit of a heterotrimeric G protein in C. elegans prevents correct spindle orientation, a process that is believed to be dependent on a Wnt and a Fz receptor, but not on Arm. Further studies regarding a possible involvement of PI-3K and G proteins in planar polarity signaling may provide additional insight to the diversity of Fz-related signaling pathways (Boutros, 1998 and references).

GENE STRUCTURE

Bases in 5' UTR - 213

Bases in 3' UTR - 519

PROTEIN STRUCTURE

Amino Acids - 623

Structural Domains

A deletional analysis of dsh has identified several conserved domains essential for activity including a so-called GLGF/DHR motif found in Drosophila discs-large(a tumor suppressor gene) and its relatives (Yanagawa, 1995), an opa repeat, and several PEST sequences (Klingensmith, 1994). The GLGF/DHR motif is also found in a vertebrate postsynaptic density protein (PSD-950) and an erythrocyte membrane protein (Klingensmith, 1994).

All Dishevelled proteins have three highly conserved domains. An N-terminal DIX (Dishevelled-Axin) domain extends for 80 amino acids and is also found in the Axin protein, which seems to be a scaffolding factor for Wnt signaling. Sequence analysis reveals that Dsh has a PDZ domain. Many other signaling proteins contain PDZ domains that can bind to a conserved stretch of amino acids at the C-termini of receptors to form homotypic complexes with other PDZ domains. Multi-domain PDZ domain proteins have been proposed to integrate signaling molecules into larger complexes. A third domain at the C-terminal part of Dsh, called the DEP (Dishevelled-EGL-10-Pleckstrin), can be found in signaling factors such as the RGS protein family, that are involved in G protein signaling. It is not yet known whether DEP domains play an active role in regulating G proteins. Although Dsh and its domains are highly conserved from Drosophila and C. elegans to mouse and human, Dsh does not have an apparent catalytic biochemical activity, nor have any clear interacting partners for Dsh been identified (Boutros, 1999).

date revised: 16 September 98 

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