Oncogene (1999) 18, 7860 ± 7872 ã 1999 Stockton Press All rights reserved 0950 ± 9232/99 $15.00 http://www.stockton-press.co.uk/onc Mechanism and function of signal transduction by the Wnt/b-catenin and Wnt/Ca2+ pathways Jerey R Miller*,1, Anne M Hocking1, Jerey D Brown1 and Randall T Moon1 1 Department of Pharmacology and Center for Developmental Biology, Howard Hughes Medical Institute, University of Washington, Seattle, Washington, WA 98195, USA Communication between cells is often mediated by activate more than one signaling pathway, we propose secreted signaling molecules that bind cell surface the names Wnt/b-catenin and Wnt/Ca2+, which receptors and modulate the activity of speci®c intracel- emphasize the downstream eectors, to describe these lular eectors. The Wnt family of secreted glycoproteins distinct signal transduction pathways. The Wnt/b- is one group of signaling molecules that has been shown catenin and Wnt/Ca2+ pathways will serve as a to control a variety of developmental processes including paradigm for elucidating the many roles Wnt genes cell fate speci®cation, cell proliferation, cell polarity and play in development and the mechanisms by which the cell migration. In addition, mis-regulation of Wnt mis-regulation of Wnt signaling lead to human cancer. signaling can cause developmental defects and is In this review, we will summarize our current under- implicated in the genesis of several human cancers. The standing of the Wnt/b-catenin and Wnt/Ca2+ path- importance of Wnt signaling in development and in ways. We have emphasized the latest advances in the clinical pathologies is underscored by the large number ®eld and pay particular attention to the molecular of primary research papers examining various aspects of mechanisms underlying each of these pathways and Wnt signaling that have been published in the past how Wnt signaling contributes to human cancer. For several years. In this review, we will present a synopsis of additional literature, we refer the reader to several current research with particular attention paid to recent reviews for information on the function of Wnt molecular mechanism of Wnt signal transduction and signaling in development (reviewed by Cadigan and how the mis-regulation of Wnt signaling leads to cancer. Nusse, 1997; Moon et al., 1997). Owing to the rapid progress in this ®eld, we apologize to those researchers Keywords: Wnt; b-catenin; cancer; signal transduction whose data may have been omitted from this review due to limitations on space. The Wnt gene family Introduction Wnt genes encode secreted signaling proteins that share Wnt genes constitute one of the major families of overall sequence identity and a conserved pattern of developmentally signi®cant signaling molecules, con- 23 ± 24 cysteine residues (Figure 1). The original trolling a variety of processes such as cell fate members of the Wnt gene family are mouse Wnt-1 speci®cation, cell migration and cell polarity (re- and Drosophila wingless (wg). Wnt-1 was identi®ed as viewed by Cadigan and Nusse, 1997). It is now clear a gene activated in response to insertion of mouse that Wnt genes are not functionally equivalent and mammary tumor virus whose over-expression in the may exert their diverse eects through activation of mammary gland contributes to tumor formation distinct intracellular signaling pathways (reviewed by (Nusse et al., 1984; van Ooyen and Nusse, 1984). wg Moon et al., 1997). Examination of Wnt gene function was identi®ed genetically and mutations in the wg locus in a variety of organisms has provided insights into the resulted in embryonic lethality and severe patterning molecular mechanics of two of these signaling path- defects (Nusslein-Volhard and Wieschaus, 1980). ways. In one pathway, Wnt proteins function through Orthologs of Wnt-1 and wg have since been found in cell surface receptors and an intracellular signaling many organisms from C. elegans to humans, with each pathway to `activate' b-catenin, a multi-functional organism possessing multiple related Wnt genes. For protein that acts in the context of Wnt signaling to example, Drosophila possesses four and mouse at least modulate transcription of speci®c target genes. In the 18 Wnt genes. A list of known Wnt genes can be found second pathway, Wnt proteins function via cell surface in Cadigan and Nusse (1997) and on the Wnt gene receptors to stimulate an increase in intracellular Ca2+ homepage (http://www-leland.stanford.edu/*rnusse/ and the subsequent activation of protein kinase C wntwindow.html). This site also includes sequence (PKC). Although it appears that dierent Wnt proteins alignments of many Wnt genes. preferentially activate one of these two pathways, this distinction is not absolute, as the activity of Wnt Reception of Wnt signals proteins likely depends on the repertoire of receptors and other co-factors present at the cell surface (He et Dierent Wnt proteins can activate distinct intracel- al., 1997; Larabell et al., 1997). Since Wnt proteins can lular responses in a variety of experimental assays. The diversity of responses seen to dierent Wnt ligands is likely due, at least in part, to the repertoire of receptors *Correspondence: JR Miller present at the cell surface. For many years the identity Wnt signal transduction JR Miller et al 7861 Figure 1 Schematic representation of the major known components of the Wnt/b-catenin pathway. Major domains are indicated. For Wnt and Frizzled: `s' indicates signal sequence; and `c' indicates conserved cysteines. The CRD domain of Frizzled is de®ned as the cysteine rich domain. The DIX domain, identi®ed in both Dishevelled and Axin, is important for homodimerization. Dishevelled also contains a PDZ domain and a DEP domain. The DEP domain has been found in proteins that regulate GTPases and in Dishevelled has been shown to be required for recruitment of Dishevelled to the plasma membrane. Axin contains a RGS domain, which has been found in proteins that regulate G-protein signaling. Binding regions for APC, GSK-3, b-catenin and PP2A on Axin are indicated. APC is comprised of the following domains: a N-terminal region required for multimerization; seven armadillo repeats; a region consisting of 15 amino acid repeats that contains a b-catenin binding site; a region consisting of 20 amino acid repeats that contains another b-catenin binding site; interspersed between the 20 amino acid repeats are SAMP repeats that are required for binding Axin; and ®nally a basic microtubule binding region. The destruction box of b-catenin contains phosphorylation sites for GSK-3 and ubiquitination sites. APC, Axin and LEF/TCF binding sites have been mapped to the armadillo repeats of b-catenin. TCF contains a nuclear localization signal (NLS). It also contains a HMG box, which is a DNA binding motif. Binding sites for b-catenin, Groucho and CtBP on TCF are indicated of Wnt receptors remained elusive, leaving a large gap Haerry et al., 1997; Lin and Perrimon, 1999; Tsuda et in our understanding of Wnt signaling. This black box al., 1999). Sulfated glycosaminoglycans are also has now been replaced with the ®nding that members required for optimal Wg activity in Drosophila cell of the Frizzled gene family can function as Wnt lines (Reichsman et al., 1996) and for Xwnt-8 activity receptors (Figure 1; Bhanot et al., 1996, 1999; Bhat, in Xenopus embryos (Itoh and Sokol, 1994). These data 1998; Kennerdell and Carthew, 1998; Muller et al., support the idea that proteoglycans are an integral part 1999; Wang et al., 1996; Yang-Snyder et al., 1996). of a Wnt receptor complex. At this time it is unclear Frizzled genes encode seven-transmembrane proteins whether proteoglycans play a role in sequestration, (Adler et al., 1990; Park et al., 1994; Wang et al., 1996) presentation or reception of Wnt signals. Further with an amino-terminal cysteine-rich domain (CRD) biochemical analyses of the Wnt reception complex that binds Wnts with high anity (Hsieh et al., 1999b). should clarify the relationship between proteoglycans Like the Wnt gene family, members of the Frizzled and Wnt signal transduction. gene family have been identi®ed in organisms with each Wnt function is also modulated extracellularly organism possesses multiple Frizzled genes. A list of through interaction with a diverse group of secreted known Frizzled genes and sequence alignments can be Wnt inhibitors. At present, these Wnt inhibitors found on the Wnt gene homepage (http:/www- include members of the secreted Frizzled-Related leland.stanford.edu/*rnusse/wntwindow.html). Protein family (called FrzB or FRP; Finch et al., Although compelling evidence exists that Frizzleds 1997; Leyns et al., 1997; Rattner et al., 1997; Wang et act as Wnt receptors, it is becoming clear that other al., 1997), Wnt-inhibitory factor-1 (WIF-1; Hsieh et al., cell surface and extracellular molecules also play 1999a), Cerberus (Bouwmeester et al., 1996; Piccolo et crucial roles in the reception of Wnt signals. In al., 1999), and Dickkopf (Fedi et al., 1999; Glinka et Drosophila, mutations in genes encoding the core al., 1998). FRP's, WIF-1, and Cerberus can directly protein of glypican, a heparan sulfate proteoglycan, bind Wnt proteins and are thought to antagonize Wnt or enzymes involved in the biosynthesis of heparan function by preventing their interaction with Frizzled. sulfate have been shown to impair wg signaling during FRP can also interact with Frizzled suggesting that embryogenesis (Binari et al., 1997; Hacker et al., 1997; FRPs may antagonize Wnt signaling through the Wnt signal transduction JR Miller et al 7862 formation of a non-functional complex with Frizzled receptors (Ba®co et al., 1999). The mechanism of Dickkopf function, however, is unknown. Interestingly, Cerberus is not only an inhibitor of Wnt, but is also an antagonist of Nodal and Bone Morphogenetic Protein (BMP) activity suggesting that it can function as a multi-valent antagonist of growth factor signaling (Piccolo et al., 1999).