Dishevelled: A Mobile Scaffold Catalyzing Development

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Wnt proteins are secreted glycoprotein ligands that regulate critical aspects of development, including cell proliferation, apoptosis, and cell fate. For those pathways downstream from the “canonical” Wnt/beta‐catenin signaling, from the “non‐canonical” or planar cell polarity (PCP), and from the Wnt‐Ca2+/cyclic guanosine monophosphate (cGMP) pathway, Wnt activation of its cellular receptor, a member of the superfamily of G‐protein–coupled receptor Frizzled family, requires both heterotrimeric G proteins and the phosphoprotein Dishevelled. Our understanding of the roles of Dishevelled proteins in development is evolving and most recent observations suggest that Dishevelled proteins act as scaffolds essential for Wnt signaling, providing docking sites for a diverse and interesting set of protein kinases, phosphatases, adaptor proteins, G proteins, and other scaffolds such as Axin. The protein–protein interactions of Dishevelled are dynamic, as is the spatial localization of this “toolbox” of signaling molecules involved in development. Much excitement awaits the elucidation of the complete set of tools in the toolbox and of the dynamic regulation of Dishevelled proteins and their interacting proteins.

Introduction

Cell signaling takes on a special significance in development. Cell biologists view the expansion in our understanding of cell signaling molecules, pathways, and networks as a central challenge to more fully understanding early development. Layering onto this challenge the task of creating a complex highly‐structured multicellular organism composed of 102–109 cells, all derived from a single cell, provokes a stark appreciation for the task confronting the developmental biologist. Discovered only 20 years ago, the Wnt family of secreted glycoproteins is known to regulate virtually all aspects of development (Cadigan, 2002). Not surprisingly, dysregulation of Wnt pathways results in disruption of normal development in humans, as well as other diseases including cancer (Moon et al., 2004). The study of the biology of Wnt signaling has been filled with surprises. Although features of the canonical or Wnt/beta‐catenin pathway controlling expression of genes regulated in a Lef/Tcf‐dependent manner have been uncovered by classical genetics, remarkably, new elements continue to be identified, such as their cellular receptors Frizzleds (cloned in 1996 [Wang et al., 1996]), co‐receptors LRP5/6 (identified in 2000 [Wehrli et al., 2000]), heterotrimeric G proteins (identified in 2001 [Liu et al., 2001]), and the prenylation of Wnt itself (discovered 2003 [Willert et al., 2003]) solving a 20‐plus‐year mystery in the challenge of isolating active Wnt ligand.

Whether it is in flies or in mice, the known proximal elements of the Wnt/beta‐catenin pathway include Wnt, LRP5/6, Frizzled‐1, heterotrimeric G proteins, and an interesting family of phosphoproteins termed Dishevelled for the manner in which Drosophila present themselves when the gene for the single member of this family in flies (Dsh) is inactivated (Klingensmith et al., 1994). Basic aspects of development, especially planar cell polarity (PCP), are disrupted in the Dishevelled mutant (Theisen et al., 1994). Embryonic lethality results from complete loss of Dishevelled in the fly (Perrimon and Mahowald, 1987), mimicking loss of the fly Wnt wingless (Wg). Incomplete loss of Dishevelled provokes other phenotypes, including disorganization of the ommatidia in the eye, and phenotypes in which structures in neighboring cells, for example, the denticles or wing hairs, are no longer properly organized with the plane of the epithelium, generating a decidedly “disheveled” or unkempt appearance (Krasnow et al., 1995). In mammals there are three members of the Dishevelled (Dvl) family, Dvl‐1, ‐2, and ‐3, products of three genes whose protein primary sequences differ significantly from one another, although all possess several landmark motifs and docking sites (Wharton, 2003). Results from numerous laboratories identifying numerous Dsh/Dvl‐associated proteins, suggest Dsh/Dvl proteins as core to multicomponent signaling complexes, and suggest a dynamic spatial localization for Dsh/Dvl. Each of these “leads” explored in this chapter requires vigorous follow‐up if we are to fully appreciate the multifaceted functions of Dsh/Dvl. To simplify the discussion of current information on the Dsh/Dvl family, the mammalian Dvl‐2 will be targeted for detailed analysis, with discussions expanding to paralogues and orthologues in the broader family.

Section snippets

Dishevelled, a Scaffold/“Toolbox” for Wnt Signaling

Inspection of primary sequences in silico suggests a key role of Dvl (i.e., multivalent docking). Three conserved domains provide the major landmarks of Dvls: a Dishevelled and Axin (DIX) binding domain at the N‐terminus; a post‐synaptic density‐95, discs‐large and Zonula occludens‐1 (PDZ) domain in the midregion of Dvl; and a Dishevelled, Egl‐10, Pleckstrin (DEP) domain located about midway between the PDZ domain and the C‐terminus of Dvl (Fig. 1). The DIX domain itself enables the possible

The Contents of the “Toolbox”

The three primary domains of Dvl scaffolds (i.e., DIX, PDZ, and DEP) have been well described and are conserved throughout metazoan evolution. Less well defined are the protein‐binding sites outside of these three domains of Dvls and the complete contents of these “toolboxes,” with many of the Dvl‐associated proteins only suspected of direct functional interactions with Dvl (Table I). The Dvl‐associated proteins that make up the contents of the toolbox can be organized into four functional

Mobile Scaffold?

Several reports provide background about Dvl localization and mobility in cells. In most instances, Dvl appears to be largely confined to the cytoplasm (Habas and Dawid, 2005). Overexpression of Frizzleds appears to provoke a relocalization of a significant amount of Dvl to the inner leaflet of the lipid bilayer (Fig. 3), presumed to reflect the association of Dvl with Frizzled or some Frizzled‐interacting protein (G protein [?], LRP5/6 [?]). In some cases, activation of cells by Wnt has led to

Major Hurdles Ahead

Many of the key unresolved questions about Dvl biology and the roles of Dvls in development will require new strategies. Mass spectrometric strategies of proteomics are powerful enough strategies with which to provide a detailed map of the Dvl molecules isolated from naive cells compared to Wnt‐activated cells. A major and formidable challenge of mass spectrometry–based strategies to analysis of Dvl is the relative low abundance of the molecules. As protein abundance in most cells spans five,

Concluding Remarks

Major gaps remain in our understanding of the molecular basis of Wnt action in development. One such high‐value target for analysis is the phosphoprotein Dishevelled, shown to play an essential role in development of the fly, worm, frog, fish, and mammals. Dvl is at the vertex of cell signaling emanated from Wnts downstream to the canonical Wnt/beta‐catenin pathway and PCP. Cell proliferation, apoptosis, and cell fate are governed largely by Wnt‐derived signals, so the gaps in our knowledge of

Acknowledgments

We would like to thank the National Institutes of Health for the generous support provided to this project from the NIDDKD (to C.C.M.) and NIGMS (to H.‐y.W.).

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