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

Je€rey R Miller*,1, Anne M Hocking1, Je€rey 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 e€ectors, to describe these lular e€ectors. 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 e€ects 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 di€erent 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 Di€erent 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 di€erent 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 anity (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).

The Wnt/b-catenin pathway

At the heart of the Wnt/b-catenin pathway is b- catenin, a multi-functional protein with independent roles in cadherin-mediated cell adhesion and Wnt signal transduction (Figure 1, reviewed by Miller and Moon, 1996; Willert and Nusse, 1998(www.stke.org)). The ultimate result of Wnt/b-catenin pathway activa- tion is the formation of a free, signaling pool of b- catenin in the cell that enters the nucleus and forms a complex with members of the LEF/TCF family of Figure 2 Schematic representation of the Wnt/b-catenin path- transcription factors to regulate expression of target way. Recent studies clearly demonstrate that Wnt/b-catenin signal genes. This is accomplished by controlling the stability transduction is not a linear progression. Instead, Wnt/b-catenin of b-catenin. In the absence of Wnt signals, b-catenin is signaling involves the regulation of a multiprotein complex. It is ubiquitinated and degraded by the proteasome path- apparent that the composition of this complex is dynamic with movement of protein in and out of the complex resulting in way, keeping the levels of free b-catenin low (Figure 2). distinct functional consequences. It is also evident that proteins Activation of the Wnt/b-catenin pathway prevents are still being identi®ed that are critical for Wnt/b-catenin ubiquitination of b-catenin and leads to the generation signaling. Therefore, we acknowledge that this schematic is a of a signaling pool of b-catenin in the cell (Figure 2). simpli®cation of the current model of the Wnt/b-catenin pathway that is discussed in detail in the text. In the absence of Wnt Gsk-3 Although this scheme appears simple, recent studies phosphorylates b-catenin. This phosphorylation is facilitated by have uncovered an increasing number of proteins that Axin and APC. Phosphorylation of b-catenin targets it for play a role in regulating b-catenin stability and the ubiquitination by b-TrCP resulting in the proteosomal degrada- mechanism by which these proteins regulate b-catenin tion of b-catenin. In the absence of Wnt, some members of TCF/ stability has proven to be a complex problem. LEF family of transcription factors act as a repressor through interaction with the co-repressors, Groucho, CBP and CtBP. In The Wnt/b-catenin pathway has been intensely contrast, in the presence of Wnt signaling, Dishevelled (Dsh) and studied in a variety of organisms and a general GBP inhibit phosphorylation of b-catenin by GSK-3. The understanding of the molecular signal transduction molecular mechanism for this inhibition is unclear but may pathway has been elucidated. Components of the Wnt/ involve either direct downregulation of GSK-3 activity or modi®cation of the composition of the complex. Consequently, b-catenin pathway were originally identi®ed as muta- a free pool of b-catenin accumulates in the cytoplasm and is now tions that produced patterning defects similar to or available to translocate it to the nucleus. Nuclear b-catenin binds opposite of those seen in wg mutant embryos (reviewed TCF/LEF and activates transcription of target genes by Cadigan and Nusse, 1997). More recently, biochemical, yeast-two-hybrid and activity-based screens have identi®ed several additional components Axin, APC, and b-TrCP/Slimb. These proteins of the pathway (Rubinfeld et al., 1993; Su et al., 1993; comprise the `destruction complex' and function to Yost et al., 1998; Zeng et al., 1997). With these maintain low steady-state levels of b-catenin in the cell. molecules in hand, a variety of approaches have been In its simplest form, Axin and APC serve as sca€olds, used to understand how these proteins regulate Wnt binding both b-catenin and GSK-3 and facilitating the signaling and order these proteins into a signal phosphorylation of b-catenin by GSK-3. GSK-3 is transduction pathway. Although the Wnt/b-catenin thought to phosphorylate one or more conserved serine pathway was once thought of as a linear pathway and threonine residues in the amino-terminal region of progressing from the plasma membrane to the nucleus, b-catenin (S33, S37, T41, and S45 in human b-catenin). these studies have shown that the pathway may be These phospho-amino acids act as a tag, promoting better described as a dynamic complex of proteins, interaction of b-catenin with b-TrCP/Slimb, a compo- coordinately acting to regulate steady-state levels of b- nent of the SCF ubiquitin ligase complex. Ubiquitina- catenin in the cell. tion of b-catenin then serves as a second tag, marking it for destruction by the proteasome. Using this model as a guide, we will examine the role each component of The destruction complex: GSK-3, Axin, APC, and the destruction complex plays in regulating steady-state b-TrCP/Slimb levels of b-catenin. The elaborate complex of proteins that, in the absence The central player in the destruction complex is of Wnt signals, promotes the ubiquitination and GSK-3, a serine/threonine kinase which is an essential subsequent degradation of b-catenin via the protea- negative regulator of the Wnt/b-catenin pathway. some is a current focus of study in the Wnt signaling Mutation of zw3, the Drosophila ortholog of GSK-3, ®eld. Presently, four proteins have been identi®ed that results in accumulation of Armadillo, the Drosophila directly promote the degradation of b-catenin: GSK-3, ortholog of b-catenin, and phenotypes that mimic Wnt signal transduction JR Miller et al 7863 constitutive activation of the Wnt/b-catenin pathway or more highly conserved serine and threonine residues (Peifer et al., 1994; Siegfried et al., 1992, 1994). near the amino-terminus of b-catenin in vitro and Similarly, over-expression of a kinase dead mutant of mutation of these putative GSK-3 sites in b-catenin GSK-3 in Xenopus promotes the stabilization and signi®cantly diminishes its phosphorylation in vitro and nuclear accumulation of b-catenin (Larabell et al., in vivo (Yost et al., 1996). These residues are part of 1997; Yost et al., 1996). GSK-3 can phosphorylate one the `destruction box' sequence that is required for

a

b

Figure 3 Mutations of b-catenin in human cancers. (a) Analysis of the location and frequency of reported b-catenin mutation in human tumors. Amino acid sequence of the destruction box of human, mouse and Xenopus b-catenin (aa32 ± 45). Numerical superscripts indicate the number of times a particular mutation has been identi®ed. Letter superscripts indicate cited reference. De3 indicates a deletion in exon 3. (b) Histogram depicting location and frequency of reported b-catenin mutations. A(Iwao et al., 1998; Morin et al., 1997; Samowitz et al., 1999; Sparks et al., 1998); B(Kobayashi et al., 1999); C(Blaker et al., 1999); D(de La Coste et al., 1998; Legoix et al., 1999; Miyoshi et al., 1998); E(Iwao et al., 1999); F(Zurawel et al., 1998); G(Gamallo et al., 1999; Palacios and Gamallo, 1998; Sagae et al., 1999; Wright et al., 1999); H(Chan et al., 1999); I(Voeller et al., 1998); J(Iwao et al., 1999); K(Fukuchi et al., 1998); L(Koesters et al., 1999); *multiple mutations were found in two samples Wnt signal transduction JR Miller et al 7864 et al., 1998; Hart et al., 1998; Kishida et al., 1998; Nakamura et al., 1998) and deletion of the RGS domain results in the production of a dominant- negative Axin protein (Zeng et al., 1997). The central region of Axin binds b-catenin and GSK-3 through distinct binding sites (Behrens et al., 1998; Hart et al., 1998; Ikeda et al., 1998; Itoh et al., 1998; Sakanaka et al., 1998; Yamamoto et al., 1998). The carboxy terminal region appears to be important for homo- dimerization (Hsu et al., 1999; Kishida et al., 1999; Sakanaka and Williams, 1999) and for interactions with Protein Phosphatase 2A (PP2A; Hsu et al., 1999) and Dishevelled (Hsu et al., 1999; Kishida et al., 1999). The proposed function of Axin is to act as a bridge between GSK-3 and b-catenin, facilitating GSK-3- dependent phosphorylation and the subsequent degra- dation of b-catenin. Consistent with this proposed role, expression of Axin in mammalian cells is sucient to reduce steady-state levels of b-catenin (Behrens et al., 1998; Hart et al., 1998; Kishida et al., 1998) and inhibits Wg signaling in Drosophila (Hamada et al., 1999; Willert et al., 1999a). Furthermore, Axin was found to promote GSK-3-mediated phosphorylation of b-catenin in vitro (Ikeda et al., 1998; Yamamoto et al., 1998). The function of Axin in down-regulating b- catenin may itself be regulated by GSK-3. Axin is a substrate for GSK-3 (Ikeda et al., 1998; Yamamoto et Figure 4 Schematic representation of the Wnt/Ca2+ pathway. al., 1998) and phosphorylation of Axin by GSK-3 can The current model for the Wnt/Ca2+ pathway is that Rat increase the stability of Axin in the cell (Yamamoto et Frizzled-2 and several mouse Frizzled homologs (Sheldahl et al., al., 1998). An additional mode of modulating Axin 1999) stimulate the release of intracellular calcium and activate activity may be through regulation of Axin/b-catenin Protein Kinase C (PKC). Recent data suggests that Frizzleds in interactions. Willert et al. (1999b) demonstrated that the Wnt/Ca2+ pathway are acting through the bg subunits of pertussis toxin sensitive G-proteins. The cellular responses in the the state of Axin phosphorylation correlated with Wnt/Ca2+ pathway are currently unknown changes in the ability of Axin to bind b-catenin. Phosphorylated Axin was found to bind b-catenin more e€ectively than dephosphorylated Axin (Willert targeted degradation. Mutation or deletion of these et al., 1999b). Lithium, a potent inhibitor of GSK-3, serine and threonine residues in the destruction box promoted the dephosphorylation of Axin providing results in highly stable forms of b-catenin that are evidence that GSK-3-mediated phosphorylation of constitutively active (Pai et al., 1997; Yost et al., 1996). Axin may play a role in modulating Axin/b-catenin The importance of the destruction box sequence is interactions (Willert et al., 1999b). highlighted by the recent ®nding that residues in the APC was originally identi®ed as a tumor suppressor destruction box of b-catenin are often mutated in a protein and mutations in APC are implicated in variety of human cancers (Figure 3, discussed in detail colorectal cancer (reviewed by Polakis, 1997). The below). APC gene encodes a large, modular protein that binds Although GSK-3 plays a crucial role in regulating b- to several components of the Wnt/b-catenin pathway, catenin stability, it does not bind b-catenin directly and including b-catenin, GSK-3, and Axin (Figure 1). requires two additional proteins, Axin and APC, to Insight into the function of APC came from the facilitate its interaction with b-catenin. Axin is the ®nding that colorectal adenocarcinoma cell lines product of the fused locus in mice and encodes a harboring mutations in the APC gene possess high modular protein that interacts with a variety of levels of b-catenin (Munemitsu et al., 1995). Expression signaling proteins including APC, b-catenin, GSK-3 of wild-type APC in these cells resulted in a dramatic and Dishevelled (Figure 1, Behrens et al., 1998; reduction in b-catenin levels suggesting that APC is Fagotto et al., 1999; Hart et al., 1998; Ikeda et al., negative regulator of b-catenin stability (Munemitsu et 1998; Itoh et al., 1998; Kishida et al., 1998; Nakamura al., 1995). et al., 1998; Sakanaka et al., 1998; Yamamoto et al., Current models predict that APC functions in a 1998; Zeng et al., 1997). Axin possesses two conserved similar fashion to Axin, facilitating GSK-3-mediated domains, an amino-terminal RGS (Regulator of G- phosphorylation of b-catenin and stimulating its protein Signaling) domain and a carboxy-terminal DIX subsequent degradation (reviewed by Moon and domain (found in Axin and Dishevelled; Cadigan and Miller, 1997; Polakis, 1997). Consistent with this idea, Nusse, 1997). RGS domains are found in a group of expression of APC in colon carcinoma cell can proteins known to regulate signaling through hetero- promote the degradation of b-catenin (Hayashi et al., trimeric G-proteins (reviewed by Dohlman and 1997; Munemitsu et al., 1995). However, over- Thorner, 1997), although there is no evidence that expression of APC in Xenopus results in phenotypes Axin interacts with G-proteins. The RGS domain, that mimic activation of the Wnt/b-catenin pathway however, is required for the binding of APC (Behrens suggesting that APC can also have a positive signaling Wnt signal transduction JR Miller et al 7865 role (Vleminckx et al., 1997). These results can be instead inhibits dephosphorylation of Wnt pathway reconciled by proposing that ectopic APC binds to components such as Axin. Further analysis of the essential negative regulators of b-catenin, such as e€ects of PP2A on the activity of the destruction GSK-3 and Axin, which may be in limiting supply. complex should help clarify the relationship between Competition for these limiting components of the PP2A and Wnt signaling. destruction complex may allow b-catenin to evade degradation and perform its signaling function. Regulation of the destruction complex and stabilization Evidence for this type of competition comes from of b-catenin Miller and Moon (1997) who found that over- expression of a membrane-tethered b-catenin resulted How does activation of the Wnt/b-catenin pathway in accumulation of endogenous b-catenin. Given these promote the stabilization and formation of a free, opposing results, further analysis of APC in a variety signaling pool of b-catenin in the cell? This question of systems should clarify the role APC plays in has been the focus of intense research over the past regulating b-catenin stability. several years and great strides have been made in our How does phosphorylation of b-catenin stimulate understanding of b-catenin regulation. At the heart of ubiquitination? The answer to this question came this question is our protagonist, b-catenin, its nemesis, recently with the ®nding that b-TrCP/Slimb, a the GSK-3/Axin/APC/b-TrCP destruction complex, component of the SCF ubiquitin ligase complex, and the Jedi Knights, Dishevelled (Dsh) and GBP/ speci®cally binds phosphorylated b-catenin (Kitagawa Frat1, which protect b-catenin from degradation. Until et al., 1999; Liu et al., 1999; Winston et al., 1999). recently, our knowledge of the molecular mechanism of Interaction of b-catenin with the SCFb-TrCP ubiquitin Dsh and GBP/Frat1 function was limited, but over the ligase complex then results in ubiquitination of b- past year signi®cant advances have been made. These catenin (Kitagawa et al., 1999; Orford et al., 1997), insights have come from examination of Wnt signaling which then targets it for proteolysis by the 26S in a variety of systems using diverse approaches and proteasome (Orford et al., 1997). Evidence for this lead to our current understanding of how Wnt signals model comes from studies examining the e€ects of b- regulate b-catenin stability. TrCP/Slimb on Wnt/b-catenin signaling in Drosophila Genetically, Dsh is the most upstream intracellular and Xenopus. Loss of slimb function in Drosophila component of the Wnt/b-catenin pathway and is results in ectopic activation of the Wnt/b-catenin activated in response to Wnt signals (Noordermeer et pathway and accumulation of high levels of Armadillo al., 1994; Siegfried et al., 1994). Dsh is a modular (Jiang and Struhl, 1998). In Xenopus, over-expression phosphoprotein that appears to function through its of b-TrCP/Slimb inhibits Xwnt-8 activity (Marikawa association with other signaling proteins (Figure 1; and Elinson, 1998) and over-expression of a dominant- Axelrod et al., 1998; Boutros et al., 1998; Li et al., negative version stabilizes b-catenin and activates Wnt/ 1999b). Dsh may modulate b-catenin stability through b-catenin target genes (Lagna et al., 1999; Liu et al., a direct interaction with the destruction complex. Dsh 1999; Marikawa and Elinson, 1998). has been shown to bind Axin and this interaction has The story of b-catenin appears to have other been proposed to result in down-regulation of GSK-3 characters, as additional regulators have entered the (Kishida et al., 1999; Li et al., 1999a; Smalley et al., fray in the past year. For example, a potential role for 1999). Consistent with this idea, Ruel et al. (1999) PP2A in regulating b-catenin stability was uncovered reported that expression of Dishevelled in Drosophila by Seeling et al. (1999). Expression of the B56a clone 8 and S2 cells results in an approximate 70% regulatory subunit of PP2A in mammalian cells led decrease in zw3/GSK-3 activity in an in vitro kinase to a decrease in steady-state levels of b-catenin and b- assay using a peptide substrate. Dishevelled expression catenin reporter gene expression. In Xenopus embryos, also stimulated serine phosphorylation of GSK-3 in S2 over-expression of B56a was found to inhibit Xwnt-8- cells suggesting that Dishevelled may regulate zw3/ induced expression of target genes. Conversely, GSK-3 activity via phosphorylation of zw3/GSK-3 by treatment of HEK293 cells with Okadaic acid, an an unidenti®ed kinase or inhibition of an unidenti®ed inhibitor of PP2A, leads to stabilization of b-catenin. phosphatase (Ruel et al., 1999). Lee et al. (1999) also Expression of B56a was also found to synergize with demonstrated that expression of Dishevelled in GSK-3 in decreasing steady-state levels of b-catenin, Drosophila clone 8 cells inhibited zw3/GSK-3- suggesting that PP2A may regulate b-catenin stability mediated phosphorylation of Tau protein. Further- through direct modulation of GSK-3 activity. How- more, Dishevelled can diminish the phosphorylation of ever, Willert et al. (1999b) recently reported that PP2A b-catenin and APC by GSK-3 in the presence of Axin appears to function as an activator of the Wnt/b- in vitro (Kishida et al., 1999). However, in this study catenin pathway. In that study, PP2A dephosphory- Dishevelled did not block the ability of GSK-3 to lated Axin in vitro, mimicking the ability of soluble phosphorylate a peptide substrate (Kishida et al., 1999) Wnt-3a to promote dephosphorylation of Axin in suggesting that Dishevelled inhibits Axin-stimulated C57MG cells. Furthermore, treatment of C57MG cells GSK-3-dependent phosphorylation of b-catenin and with Okadaic acid inhibited Wnt-3a, and resulted in an APC via a mechanism other than a direct e€ect on increase in the association of b-catenin with Axin and a GSK-3 kinase activity itself. decrease in b-catenin stability. There are several Dsh function may also be dependent on its explanations for these discrepancies. First, PP2A is a localization within the cell. In Xenopus, the activation multi-protein enzyme and the speci®c composition of of a maternal Wnt/b-catenin pathway is required for subunits may alter its function depending on the the establishment of dorsal cell fates (reviewed by cellular context. It is also possible that over-expression Moon and Kimelman, 1998). Examination of the of the B56a regulatory subunit does not promote, but localization of Dsh in early embryos revealed that Wnt signal transduction JR Miller et al 7866 Dsh associates with small vesicle-like organelles (0.5 ± conditioned media causes a twofold reduction in 1.0 mm diameter) that are enriched on the dorsal side GSK-3 activity via a PKC-dependent mechanism. of the embryo at the end of the ®rst cell cycle (Miller et Likewise, stimulation of the Wnt/b-catenin pathway al., 1999). This localization appears to be signi®cant in Drosophila clone 8 cells via addition of soluble Wg, since treatments that prevent dorsal development also or expression of DFz-2 caused a decrease in zw3/GSK- block the dorsal enrichment of Dsh. Interestingly, time- 3 activity in cell extracts (Ruel et al., 1999). GSK-3 lapse confocal microscopy analysis of Green Fluor- activity can also be modulated by Wnt signals in vivo. escent Protein tagged Dsh (Dsh-GFP) localization in Itoh et al. (1998) have demonstrated that over- early embryos demonstrated that Dsh-GFP associates expression of Xwnt-8 in Xenopus embryos results in a with and is transported along the microtubule decrease in GSK-3 activity associated with Axin. This cytoskeleton towards the prospective dorsal side of e€ect was not due to modulation of Axin/GSK-3 the embryo. Dsh also localizes to small vesicle-like interactions since expression of Xwnt-8 did not alter organelles in both mammalian and Drosophila cell lines the levels of GSK-3 associated with Axin. Over- (Lee et al., 1999; Smalley et al., 1999). Together these expression of Wg in early Drosophila embryos also data suggest that regulation of Dsh localization, resulted in a modest decrease in total zw3/GSK-3 perhaps through its association with the cytoskeleton, activity (Ruel et al., 1999). Together, these results plays an important role in modulating the activity of demonstrate that Wnt signals can inhibit GSK-3 the Wnt/b-catenin pathway. activity. GBP (GSK-3 Binding Protein), originally identi®ed Although a case can be made for the importance of in a yeast-two-hybrid screen using GSK-3 as bait, and GSK-3 down-regulation in controlling b-catenin its mammalian ortholog Frat1 function as positive stability, several recent papers provide evidence that regulators of the Wnt/b-catenin pathway. In Xenopus, Wnt signals do not inhibit GSK-3 per se, but instead over-expression of GBP/Frat1 mimics activation of the alter the composition of the destruction complex Wnt/b-catenin and results in formation of an ectopic thereby suppressing the phosphorylation of b-catenin dorsal-ventral axis (Yost et al., 1998). Expression of by GSK-3. Willert et al. (1999) showed that Wnt-3a GBP also results in stabilization of b-catenin. Insight stimulation of C57MG cells results in a reduction in into the mechanism of GBP function come from data the phosphorylation of Axin that coincides with a showing that GBP antagonizes GSK-3-dependent decrease in binding anity between Axin and b- phosphorylation of Tau both in vitro and in vivo, catenin. Li et al. (1999) found that expression of suggesting that it stabilizes b-catenin through inhibition Wnt-1 promotes the disintegration of the Axin-GSK-3- of GSK-3 activity. However, it is unclear whether this Dsh-GBP/Frat1 complex in cultured cells. A GSK-3 e€ect is due to a change in GSK-3 activity or through binding peptide from GBP/Frat1 was also shown to steric blockade of GSK-3/substrate interactions. prevent GSK-3 interactions with Axin and GSK-3- It is possible that Dishevelled and GBP/Frat1 mediated phosphorylation of Axin and b-catenin function independently, but recent evidence suggests (Thomas et al., 1999). Finally, Ruel et al. (1999) that their roles in regulating b-catenin stability may be reported that addition of soluble Wg to Drosophila intertwined. Dishevelled and GBP/Frat1 interact in clone 8 cells not only suppressed the activity of zw3/ mammalian cells and Xenopus embryos (Li et al., GSK-3 but also caused a decrease in its interaction 1999a). Furthermore, Dishevelled and GBP/Frat1 were with Axin. Thus, Wnt signals can also modulate the found in a quaternary complex with GSK-3 and Axin composition of the destruction complex thereby (Li et al., 1999a). Interestingly, composition of this preventing GSK-3-dependent phosphorylation of b- complex is regulated by Wnt-1 expression suggesting catenin. Further biochemical analysis of the destruction that interactions between GSK-3, Axin, Dishevelled, complex and its regulation by Dsh and GBP/Frat1 will and GBP/Frat1 may be important for regulating b- help resolve how Wnt signals promote the stabilization catenin. of b-catenin in the cell. Current models predict that Wnt-mediated activa- tion of Dsh and GBP/Frat1 inhibits GSK-3-mediated Regulation of b-catenin stability by other signaling phosphorylation of b-catenin thereby stimulating the pathways signaling function of b-catenin. Although this hypoth- esis is generally agreed upon, there is some debate b-catenin can also be activated by Wnt-independent concerning the mechanism by which Dsh and GBP/ mechanisms. Expression of Integrin-linked kinase Frat1 modulate GSK-3 function. Two alternative (ILK) in mammalian cells promotes the stabilization hypotheses have been proposed to explain how and nuclear accumulation of b-catenin (Novak et al., activation of Dsh and GBP/Frat1 lead to down- 1998). ILK can phosphorylate and down-regulate regulation of GSK-3-mediated phosphorylation of b- GSK-3 activity (Delcommenne et al., 1998) suggesting catenin: (1) Activation directly down-regulates GSK-3 that its e€ects on b-catenin may result from the activity, or (2) Activation alters the composition of the inactivation of GSK-3. Presenilins have also been destruction complex thereby preventing GSK-3 phos- implicated as regulators of b-catenin stability (Kang phorylation of b-catenin. Although these proposed et al., 1999; Tesco et al., 1998; Zhang et al., 1998). mechanisms are not mutually exclusive and cell type- Mutations in presenilin associated with rapid onset of speci®c di€erences may exist, they serve as a paradigm Alzheimer's disease decrease the stability of b-catenin for understanding the mechanism of signal transduc- in neurons (Zhang et al., 1998). This e€ect on b-catenin tion through the Wnt/b-catenin pathway. was also correlated with an increase in the suscept- Several groups have shown that Wnt signals can ibility of neurons to apoptosis resulting from the inhibit GSK-3 activity. Cook et al. (1996) have shown accumulation of b-amyloid protein (Zhang et al., that stimulation of mammalian cells with Wg 1998). Given the ability of these, and other, signaling Wnt signal transduction JR Miller et al 7867 pathways to modulate b-catenin stability, it is likely pathway in C. elegans (Meneghini et al., 1999; that b-catenin may regulate many diverse cellular Rocheleau et al., 1999) and the ability of TAK-1 and processes independent of Wnt signaling. NLK to regulate b-catenin/TCF-function in vertebrates (Ishitani et al., 1999). These studies provide evidence that TAK-1 (MOM-4) stimulates NLK (LIT-1) activity Downstream of b-catenin: targets and regulation (Ishitani et al., 1999). WRM-1, a C. elegans ortholog of In the presence of Wnt signals, b-catenin is stabilized b-catenin/Armadillo (Rocheleau et al., 1997), also and accumulates both in the cytoplasm and the nucleus interacts with and activates NLK/LIT-1 (Rocheleau (Peifer et al., 1994; Yost et al., 1996). In the nucleus, b- et al., 1999). Active NLK/LIT-1 then phosphorylates catenin binds members of the TCF/LEF family of TCF/POP-1 (Ishitani et al., 1999; Rocheleau et al., transcription factors (Figure 1, reviewed by Cavallo et 1999) and prevents TCF/POP-1 from binding DNA al., 1997) and this complex stimulates expression of (Ishitani et al., 1999). In vertebrates and Drosophila, Wnt/b-catenin target genes. A number of target genes this would block the ability of b-catenin/TCF to have been identi®ed in the past several years and these activate transcription, whereas in C. elegans this include developmental regulatory genes such as would interfere with the ability of POP-1 to repress siamois, twin,andXnr-3 in Xenopus (Brannon et al., transcription. Further analyses of TAK-1/MOM-4 and 1997; Fan et al., 1998; Laurent et al., 1997; McKendry NLK/LIT-1 function will help clarify how other et al., 1997), and ultrabiothorax in Drosophila (Riese et growth-factor-mediated signaling pathways converge al., 1997). Additional targets include regulators of cell with the Wnt signaling pathway to modulate diverse growth and proliferation, c-myc and cyclin D-1 (He et cellular processes during development. al., 1998; Shtutman et al., 1999; Tetsu and McCormick, 1999), a regulator of cell ± cell communication, con- Oncogenic activation of the Wnt/b-catenin pathway nexin-43 (van der Heyden et al., 1998), and the metalloproteinase, matrilysin (Crawford et al., 1999). Researchers have long recognized the similarities The mechanism by which b-catenin activates between the growth and di€erentiation of cells and transcription remains unclear. Mutational analyses tissues during development and the mis-regulation of have identi®ed two regions of b-catenin, one near the these events in oncogenesis. This relationship has amino-terminus and one at the carboxy-terminus, that become more evident with the observation that many are important for transactivation (Cox et al., 1999; proto-oncogenes encode components of growth factor- Hecht et al., 1999; van de Wetering et al., 1997). mediated signal transduction pathways. The Wnt genes Another possible mode of action is that b-catenin and components of intracellular Wnt signaling path- binding to TCF/LEF may displace co-repressors (see ways provide one of the most striking examples of this below) from TCF/LEF. At this time, however, it is not connection. In particular, the discovery that activating known whether association of b-catenin and one of mutations in b-catenin are associated with a variety of several identi®ed co-repressors with TCF/LEF is human cancers has fueled an extraordinary explosion mutually exclusive. of interest into the relationship between Wnt signaling In the absence of Wnt signals, TCF/LEF proteins and oncogenesis. can act as transcriptional repressors, preventing Wnt-1 was originally identi®ed as a preferred transcription of Wnt/b-catenin target genes. Evidence integration site for mouse mammary tumor virus in supporting this proposal came from examination of the breast adenocarcinomas (Nusse and Varmus, 1982). In transcriptional control of siamois in Xenopus and ubx addition, Wnt-1 is able to transform cultured in Drosophila (Brannon et al., 1997; Riese et al., 1997). mammary epithelial cell lines, C57MG and RAC Mutation of the TCF/LEF binding sites in siamois and (Brown et al., 1986; Rijsewijk et al., 1987) and ubx reporter constructs resulted in a moderate transgenic mice expressing Wnt-1 in the mammary activation of the genes in the absence of Wnt signals. gland develop mammary epithelia hyperplasias and TCF/LEF proteins do not appear to act as repressors adenocarcinomas (Edwards et al., 1992; Tsukamoto et on their own but require interactions with one of al., 1988). However, in these transgenic studies, it is several recently identi®ed co-repressors including clear that Wnt-1 expression alone is insucient for Groucho (Cavallo et al., 1998; Roose et al., 1998), malignant transformation and probably requires other CBP/p300 (Creb Binding Protein; Waltzer and Bienz, genetic defects. Although these data make a case for 1998), and CtBP (C-terminal Binding Protein; Brannon Wnt-1 acting as an oncogene, mutations in Wnt-1 have et al., 1999). The mechanism by which Groucho and not been linked to cancer in humans. However, since CtBP repress transcription is unclear. However, mutations in several components of the Wnt/b-catenin Waltzer and colleagues (1999) demonstrated that CBP pathway are implicated in tumor formation it is likely acetylates dTCF in vitro and that this results in a that Wnt-1, or other Wnt proteins capable of modest reduction in dTCF/Armadillo binding. These activating the Wnt/b-catenin pathway, may act as data suggest that CBP may function by modulating oncogenes in humans. interactions between TCF/LEF and b-catenin raising Germline mutations of the APC gene lead to familial the threshold level of b-catenin required for activation adenomatous polyposis (FAP) characterized by the of target genes. development of colorectal polyps in the second to third Recent evidence also implicates components of the decade of life (Groden et al., 1991; Nishisho et al., mitogen-activated protein kinase (MAPK) pathway, 1991). In addition, somatic mutations of the APC gene transforming growth factor-b-activated kinase (TAK-1) are associated with 480% of sporadic colorectal and NEMO-like kinase (NLK), as antagonists of TCF adenomas and carcinomas (Miyoshi et al., 1992; function. Evidence for the involvement of TAK-1 and Powell et al., 1992; Smith et al., 1993). Greater than NLK come from genetic analyses of a Wnt signaling 95% of germline and somatic mutations of the APC Wnt signal transduction JR Miller et al 7868 gene are nonsense mutations in the 5' half of the GSK-3 sites in the epidermis of transgenic mice coding sequence that result in the formation of resulted in the formation of hair follicle-related tumors truncated protein products (reviewed by Polakis, (Gat et al., 1998). Cell lines harboring activating 1997). The region in which these mutations accumu- mutations of b-catenin often display high levels of late has been termed the mutation cluster region both cytoplasmic and nuclear b-catenin and constitu- (MCR). The ®nding that the APC protein binds and tive activation of TCF/LEF reporter constructs regulates the steady-state levels of b-catenin ((Mune- (Korinek et al., 1997). These data demonstrate that mitsu et al., 1995; Rubinfeld et al., 1993; Su et al., increasing levels of b-catenin is sucient to promote 1993) discussed above) provide the ®rst insights into tumor formation and implicate the amino-terminal how these mutations contribute to tumor formation. In GSK-3 sites as potential targets for the oncogenic colorectal adenocarcinoma cells expressing mutant activation of b-catenin. APC, b-catenin was found to accumulate to high Recent studies demonstrate that b-catenin is highly levels and expression of wild-type APC results in a mutable in various types of cancer and highlight the dramatic reduction in b-catenin levels (Munemitsu et relationship between regulation of b-catenin stability al., 1995). Signi®cantly, structure-function studies and oncogenesis. Figure 3a provides a list of reported demonstrated that the central third of APC is mutations in b-catenin associated with human cancers sucient and required for down-regulation of b- and the tissue from which each mutant was isolated catenin and mutant APC proteins truncated at the (see Figure 3 legend for a complete list of references). MCR are incapable of promoting b-catenin degrada- Strikingly, the majority of the identi®ed mutations are tion (Munemitsu et al., 1995). This central region of missense mutations in one of the putative GSK-3 APC contains binding sites for b-catenin and Axin as phosphorylation sites (Figure 3b). Missense mutations well as putative phosphorylation sites for GSK-3 at D32 and G34 have also been reported. The DSG (Figure 1, (Rubinfeld et al., 1996)) and lack of these sequence along with S37 has been characterized as a sites is thought to explain the abnormal function of ubiquitination target motif based on its similarity with truncated APC proteins. Ik-B, another protein targeted for degradation by the Introducing speci®c mutations into the murine APC SCFb-TrCP ubiquitin ligase complex (Chen et al., 1995, gene has generated several mouse models of APC 1996; Winston et al., 1999). In addition to missense function. Deletion of the APC gene results in mutations, a number of deletion mutants that remove embryonic lethality prior to day 8 of gestation one or more of the putative GSK-3 sites have been demonstrating a requirement for APC during devel- isolated. Together, these data strongly argue that opment (Fodde et al., 1994; Moser et al., 1995; mutations in b-catenin that enable it to evade Oshima et al., 1995). Heterozygous Min (Multiple regulation by the destruction complex play an intestinal neoplasia) mice, which harbor a germline important role in the tumorigenic transformation of mutation in APC, develop a severe intestinal many cell types. phenotype characterized by the formation of 4100 What is the consequence of constitutive b-catenin small intestinal tumors (Moser et al., 1992; Su et al., activity? This question remains unanswered, but 1992). APCD716 mice also develop a large number of several recent papers provide some insight into the intestinal tumors (Oshima et al., 1995). Both of these role b-catenin plays in tumorigenesis. Several groups alleles encode truncated forms of APC that lack the demonstrated that the c-myc and cyclin D1 genes, central region required for b-catenin regulation. A both of which are known to promote cell prolifera- conditional mutant has also been generated to tion, are direct targets of b-catenin (He et al., 1998; produce a truncated form of APC and upon Shtutman et al., 1999; Tetsu and McCormick, 1999). recombination mice developed multiple colorectal c-myc is a potent oncogene that regulates cell cycle

adenomas within 4 weeks (Shibata et al., 1997). In progression, promoting the G1/S phase transition. addition to the well-documented link between APC However, c-myc cannot transform cells on its own function and colorectal cancer, mutation of APC is and requires survival cues to prevent cells from also implicated in aggressive ®bromatosis (Alman et undergoing apoptosis (reviewed in Steiner, 1996; al., 1997; Li et al., 1998) and as a genetic modi®er of Thompson, 1998). Thus, b-catenin may also provide BRCA penetrance for breast cancer (Redston et al., cell survival signals that protect the cell from 1998). Consistent with this latter ®nding, Min mice are apoptosis. Consistent with this idea, Orford and susceptible to mammary gland tumorigenesis (Bilger et colleagues (1999) demonstrated that expression in al., 1996). Together these ®ndings highlight the MDCK cells of wild-type b-catenin or a form of b- importance of APC in normal development and catenin harboring an S37A missense mutation resulted oncogenesis. The use of mouse models of APC in a signi®cant increase in proliferation, and protec- formation will provide tools for not only investigating tion from anoikis (suspension-induced apoptosis). In

how mutation of APC promotes tumor formation but addition, b-catenin expression attenuated the G1 to S also for developing potential therapeutic agents. cell cycle block that occurs in response to DNA The striking connection between APC-mediated damage (Orford et al., 1999). Thus, mutations in b- regulation of b-catenin and oncogenesis suggested catenin that result in a more stable form of the that mutations in b-catenin itself might play a role in protein likely lead to mis-expression of target genes, tumor formation. Consistent with this idea, targeted such as c-myc and cyclin D1. Expression of c-myc and

mutation or deletion of the putative GSK-3 sites leads cyclin D1 would then expedite the G1 to S transition. to the production of highly stable forms of b-catenin Additionally, b-catenin may act as a survival factor that are hyperactive (Pai et al., 1997; Yost et al., 1996). protecting cells from cell death. Since the transition

In addition, expression of an amino-terminally from G1 to S phase requires the presence of survival truncated form of b-catenin lacking the putative factors, b-catenin may also stimulate this transition Wnt signal transduction JR Miller et al 7869 directly by preventing apoptosis and permitting cell this change can be partially blocked by expression of cycle progression. Elevated b-catenin may also Wnt-5a (Olson and Gibo, 1998). Conversely, transfec- enhance the accumulation of additional mutations by tion of parental C57MG cells with anti-sense Wnt-5a preventing the DNA damage-induced cell cycle block. constructs results in cells with a morphology similar to that seen following transformation by Wnt-1 (Olson and Gibo, 1998). In Xenopus, co-expression of Xwnt-5a with Wnt/Ca2+ pathway Xwnt-8 blocks the ability of Xwnt-8 to induce a secondary dorsal-ventral axis (Torres et al., 1996). Frizzleds can discriminate between di€erent Wnt These data suggest that one function of Wnt-5a may be ligands and this selectivity appears to be important to modulate the activity of the Wnt/b-catenin pathway. for determining which downstream pathway is Although the mechanism of Wnt-5a action is unclear, it activated in the cell. For example, expression of Rat is tempting to speculate that antagonism of the Wnt/b- Frizzled-1 (RFz-1), but not Rat Frizzled-2 (RFz-2), can catenin pathway occurs via activation of the Wnt/Ca2+ promote the accumulation of Xwnt-8 at the cell surface pathway. In support of this hypothesis, activation of and activate Wnt/b-catenin target genes (Sheldahl et Ca2+ signaling in Xenopus by ectopic expression of the al., 1999; Yang-Snyder et al., 1996). Conversely, serotonin receptor can block activation of the Wnt/b- expression of Xwnt-5a or RFz-2, but not Xwnt-8 or catenin pathway by Xwnt-8 (Slusarski et al., 1997b). RFz-1, leads to an increase in Ca2+ ¯uxes in zebra®sh, Further analyses of Wnt-5a function should help clarify and activation of PKC in Xenopus (Sheldahl et al., the relationship between the Wnt/b-catenin and Wnt/ 1999; Slusarski et al., 1997a,b). Using the induction of Ca2+ pathways. Wnt/b-catenin target genes and activation of PKC in The ability of Wnt-5a to suppress transformation by Xenopus as assays for Frizzled activity, Sheldahl et al. Wnt-1 suggests that Wnt-5a may act as a tumor (1999) demonstrated that there are two functionally suppressor. Consistent with this proposed role, ectopic distinct classes of Frizzled proteins. Expression of RFz- expression of Wnt-5a in a human renal cell carcinoma 1, mouse Frizzled-7 and -8, Xenopus Frizzled-1, and cell line, RCC233, results in suppression of cell growth Drosophila Frizzled and DFrizzled-2 activated Wnt/b- and a decrease in telomerase activity (Olson et al., 1998). catenin targets, but not PKC. On the other hand, In contrast, telomerase activity is increased signi®cantly expression of RFz-2, mouse Frizzled-3, -4, and -6 in Wnt-1 induced tumors (Broccoli et al., 1996). Stable activated PKC but not Wnt/b-catenin targets. Based on transfection of Wnt-5a into a human uroepithelial cell these results, we propose the name, Wnt/Ca2+ pathway, line, MC-T116 resulted in a loss of tumor formation in to denote the non-canonical vertebrate Wnt pathway athymic nude mice and a loss of anchorage-independent that activates Ca2+ release and PKC (Figure 4). cell growth in soft agar (Olson et al., 1997). Although the mechanism by which Frizzleds trans- Does Wnt-5a function as a tumor suppressor in vivo? duce Wnt signals is unclear, recent evidence suggests that Wnt-5a levels are not unilaterally repressed in all human RFz-2, and other Frizzleds that activate the Wnt/Ca2+ cancers. Although Wnt-5a mRNA levels are decreased in pathway, may function through activation of hetero- endometrial carcinoma compared to normal tissue (Bui trimeric G-proteins (Sheldahl et al., 1999; Slusarski et al., et al., 1997), Wnt-5a levels are up regulated in breast 1997a). The ability of RFz-2 to increase Ca2+ ¯uxes in cancer (Iozzo et al., 1995; Lejeune et al., 1995), lung zebra®sh and activate to PKC in Xenopus is sensitive to carcinoma, metastatic melanoma, and colon carcinoma pertussis toxin, suggesting that G-proteins of the Gai or (Iozzo et al., 1995). Furthermore, removal of Wnt-5a 2+ Gao class function downstream RFz-2 in the Wnt/Ca function in mice results in a decrease in cell proliferation pathway. In addition, RFz-2-mediated activation of suggesting a positive role for Wnt-5a in the regulation of PKC is blocked by expression of a-transducin, a cell growth and proliferation. Therefore, current in vivo treatment that is predicted to sequester bg subunits of evidence would appear to con¯ict with the concept of heterotrimeric G-proteins, preventing them from acti- Wnt-5a as a tumor suppressor. However, until the vating downstream e€ectors (Sheldahl et al., 1999). mechanics of Wnt-5a signal transduction is understood Consistent with the idea that some Frizzleds signal via a in vivo, the role of Wnt-5a in carcinogenesis will remain G-protein-dependent mechanism, Frizzled proteins unclear. share overall sequence similarity and predicted structure with many other G-protein coupled receptors (Barnes et al., 1998). Although a compelling case can be made for Summary the involvement of G-proteins in Frizzled-mediated signaling, it remains unclear whether G-proteins play a Over the past several years, great strides have been direct or indirect role in the Wnt/Ca2+ pathway. Further made in our understanding of the mechanics and analysis of Frizzled/G-protein interactions using chi- functions of Wnt signaling. In particular, recent studies meric inducible-Frizzled proteins or soluble Wnt should examining the relationship between Wnt signaling, the help clarify the relationship between Frizzleds and G- regulation of b-catenin stability, and oncogenesis proteins. highlight the importance of understanding how Wnt signals modulate di€erent cellular processes. However, many questions remain unanswered. How do Frizzleds Distinct roles for Wnt-5a in cell proliferation and tumor transduce Wnt signals? How do Wnt signals regulate suppression? the destruction complex? How does b-catenin activate A number of studies provide evidence that Wnt-5a is able transcription? What cellular processes does the Wnt/ to antagonize the Wnt/b-catenin pathway. Stable Ca2+ pathway regulate? With the dramatic increase in expression of Wnt-1 in C57MG mouse mammary the number of researchers examining these and other epithelial cells results in altered cell morphology and questions, answers will not be far away. Wnt signal transduction JR Miller et al 7870 Acknowledgments signal transduction in our lab is supported by the Howard The authors would like to thank members of the Moon lab Hughes Medical Institute. for helpful comments on the manuscript. Work on Wnt

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