Cell. Signal. Vol. 11, No. 11, pp. 777–788, 1999 ISSN 0898-6568/99 $ – see front matterCopyright 1999 Elsevier Science Inc. PII S0898-6568(99)00054-6

TOPICAL REVIEW

Roles of Axin in the Wnt Signalling PathwayAkira Kikuchi*

Department of Biochemistry, Hiroshima University School of Medicine,1-2-3, Kasumi, Minami-ku, Hiroshima 734-8551, Japan

ABSTRACT. The Wnt signalling pathway is conserved in various species from worms to mammals, and playsimportant roles in development, cellular proliferation, and differentiation. The molecular mechanisms by whichthe Wnt signal regulates cellular functions are becoming increasingly well understood. Wnt stabilizes cytoplasmicb-catenin, which stimulates the expression of genes including c-myc, c-jun, fra-1, and cyclin D1. Axin, newlyrecognized as a component of the Wnt signalling pathway, negatively regulates this pathway. Other componentsof the Wnt signalling pathway, including Dvl, glycogen synthase kinase-3b, b-catenin, and adenomatous polypo-sis coli, interact with Axin, and the phosphorylation and stability of b-catenin are regulated in the Axin com-plex. Thus, Axin acts as a scaffold protein in the Wnt signalling pathway, thereby regulating cellular func-tions. cell signal 11;11:777–788, 1999. 1999 Elsevier Science Inc.

KEY WORDS. Wnt, Axin, b-catenin, GSK-3b, Dvl, APC, Scaffold protein

INTRODUCTION mologue Tcf/Lef family are involved in transduction of thevertebrate Wnt signalling. These components and possibleWnt proteins constitute a large family of cysteine-rich se-signalling pathways have been proposed based on geneticcreted ligands that control development in organisms rang-evidence. Therefore, several questions must be answered toing from nematode worms to mammals [1]. At the cellularunderstand the biochemical relationship between the com-level, Wnts regulate proliferation, morphology, motility,ponents in the Wnt signalling pathway. For example, howand fate. The outlines of the Wnt signal-transduction path-does Dvl (Dishevelled) antagonize GSK-3b? Does GSK-3bway were first elucidated by a genetic analysis of Winglessphosphorylate b-catenin directly? How does the Wnt signalsignalling during the development of segmental polarity ingo specifically to b-catenin? To address these questions,Drosophila, and extended through studies of embryonic axisidentification of factors that interact with the identifiedformation in Xenopus (Fig. 1). A number of components ofcomponents of the pathway will lead to new discoveries andthe Wingless pathway have been identified [2–4]. In theinsights. Axin, recently shown to be a Wnt signal negativecurrent model, the serine/threonine kinase Zw3/shaggy tar-regulator, provides clues for answering the questions posedgets cytoplasmic Armadillo protein for degradation in theabove. In this review, the functions and roles of Axin in theabsence of Wingless. As a result, cytoplasmic ArmadilloWnt signalling pathway are described.levels are low. In the presence of Wingless, Dishevelled, a

cytoplasmic protein with unknown functions, is activated,presumably via interaction of Wingless with a cell-surface

IDENTIFICATION OF AXINreceptor, Frizzled. Dishevelled antagonizes the action ofFusedZw3, perhaps by modulating its enzymatic activity. Arma-Axin has been originally identified as the product of thedillo is no longer degraded, resulting in its accumulation in

both cytoplasm and nucleus, where it binds to pangolin, a mouse gene called Fused [5]. The Fused allele (AxinFu) andtranscription factor, and stimulates gene expression. Many two other spontaneous alleles, Kinky (AxinKi) and Knobblyelements of the Wingless pathway are shared by multi-cellu- (AxinKb), cause similar dominant phenotypes characterizedlar animals (Fig. 1). In Xenopus and mammals, the Frizzled by kinking and shortening of the tail, and also cause reces-homologue frizzled, the Dishevelled homologue Dvl, the sive embryonic lethality at E8–E10 [6–8]. Further, AxinTgl

Zw3 homologue glycogen synthase kinase-3b (GSK-3b), induced by a random transgene insertion, has no dominantthe Armadillo homologue b-catenin, and the pangolin ho- effect but caused recessive lethal embryonic defects similar

to those observed in AxinKi and AxinKb embryos [9]. Embryos*Author to whom all correspondence should be addressed. Tel.: 181-82- homozygous for the recessive lethal alleles show frequent257-5130; fax: 181-82-257-5134; e-mail: akikuchi@mcai.med.hiroshi neuroectodermal defects, including truncation or incom-ma-u.ac.jp

Received 26 may 1999; and accepted 26 July 1999. plete closure of the anterior neural folds, as well as cardiac

778 A. Kikuchi

FIGURE 1. Wnt signalling pathway.

defects. An intriguing feature of many homozygous axin same screening, an Axin homologue, Axil (Axin-like), hasbeen also identified [11]. Axil shares 44% identity withmutant embryos is a duplication of the embryonic axis [7,

8], suggesting that Axin normally plays a negative regula- Axin (Fig. 2). They are ubiquitously expressed in varioustissues including brain, thymus, heart, lung, liver, spleen,tory role in the response to an axis-inducing signal. Indeed,

when Axin is injected into Xenopus embryos, most embryos kidney, testis, and skeletal muscle. Another Axin homo-logue, conductin, has been identified as a b-catenin-bind-develop with strong axial defects [5]. Co-expression of Axin

inhibits the induction of the secondary dorsal axis by Wnt, ing protein by yeast two-hybrid screening of a mouse cDNAlibrary [12]. Conductin and Axil are the same protein, im-Dvl, and kinase-negative GSK-3b, while it does not affect

b-catenin- and Siamois-induced secondary axis formation. plying that there are at least two Axin family members inmammals. The Axin gene is conserved in humans, rats,Although injection of Noggin or dominant-negative bone

morphogenetic protein receptor also causes secondary axis mice, chickens, and Xenopus [5, 10, 13]. A homologue ofthe vertebrate Axin has also been found in Drosophila, andformation, Axin fails to block this induction [5]. These re-

sults indicate that Axin exerts its function on axis forma- named D-Axin [14]. When the amino acid numbers ofAxin are indicated in this review, they refer to rat Axintion by specifically inhibiting the Wnt signalling pathway.

However, no biochemical information has been obtained (rAxin).on how and with which components of the Wnt pathwayit might interact.

Structure of Axin

There are alternative Axin splicing products, termed formsAxin Family 1 and 2. Form 2 is identical to form 1, except for an inser-

tion of 36 amino acids at position 736 in rAxin. Axin hasIndependent biochemical approaches have demonstratedthe existence of Axin in various species. By yeast two- several unique regions (Fig. 3). The N-terminal region of

rAxin, residues 89–216, shares 31% amino acid identityhybrid screening of a rat brain cDNA library, Axin has beenidentified as a protein that binds to GSK-3b [10]. In the with residues 58–178 of RGS4, which has been identified as

Axin in the Wnt Signalling Pathway 779

FIGURE 2. Amino acid se-quences of rAxin and Axil.Identical residues in rAxin andAxil are denoted by a blackbackground. The RGS and DIXdomains are boxed.

BINDING PARTNERS OF AXINa GTPase-activating protein (GAP) for heterotrimericGSK-3bGTP-binding protein (G protein) [15]. The C-terminal-

region residues 757–820 share 37% amino acid identity As described above, Axin is a GSK-3b-binding protein.with residues 8–73 of the mouse Dsh homologue, Dvl-1 GSK-3 has been originally characterized as a serine/threo-[16]. The former and the latter regions are named the RGS nine kinase that phosphorylates and inactivates glycogenand the DIX domains, respectively. The central region of synthase and was subsequently shown to be identical to pro-Axin contains the GSK-3b- and b-catenin-binding sites, tein kinase FA, which activates ATP-Mg-dependent type-1and residues 353–437 and 437–506 are responsible for bind- protein phosphatase [19]. GSK-3 is now implicated in theing to GSK-3b and b-catenin, respectively [10]. The region regulation of several physiological responses in mammalianbetween the b-catenin-binding site and the DIX domain cells by phosphorylating many substrates, including neu-(rAxin-(506–713)) interacts with Dvl [17]. The C-termi- ronal cell adhesion molecule, neurofilament, synapsin I,nal-half region binds to protein phosphatase 2A (PP2A) tau, transcription factors, and the adenomatous polyposis[18, 89]. The site phosphorylated by GSK-3b is adjacent to coli (APC) gene product [19–22]. The cDNAs of mamma-

lian GSK-3a and GSK-3b have been isolated, and they en-the N-terminal region of the GSK-3b-binding site [10].

780 A. Kikuchi

FIGURE 3. Structure of Axin and its binding proteins. Axin possesses binding sites for APC, GSK-3b, b-catenin, Dvl, and PP2A. Inthe Axin complex, GSK-3b efficiently phosphorylates b-catenin, APC, and Axin itself, while Dvl and PP2A prevent the phosphoryla-tions.

code protein kinases with molecular masses of 51 kDa and b-catenin by GSK-3b, consequently downregulating thelevel of b-catenin.47 kDa, respectively [23]. Although both GSK-3a and

GSK-3b form complexes with Axin [10], GSK-3b is mainly Plakoglobin has been shown to interact with the cyto-plasmic regions of desmoglein and desmocollin, transmem-used in the analyses of the Wnt signalling pathway, as GSK-

3b but not GSK-3a rescues the phenotype of the Drosophila brane components of desmosomes [27–29]. The cytoplasmiccomponents of desmosomes are necessary for linking to in-zw3/shaggy gene product [24]. GSK-3b binds directly to

amino acids 353–437 of rAxin (rAxin-(353–437)) (Fig. 3). termediate filaments [30]. Plakoglobin shares 66% overallidentity with b-catenin. Both proteins have Armadillo re-GSK-3bK85M and GSK-3bY216F, in which ATP-binding and

tyrosine-phosphorylation sites are mutated, respectively, are peats in the central region, and the identity of their Arma-dillo repeats is 77%. rAxin also binds directly to Armadillocatalytically inactive mutants, and neither of them interacts

with rAxin. These results suggest that the interaction of repeats 2–7 of plakoglobin [90]. b-catenin inhibits theinteraction of plakoglobin with rAxin, suggesting thatGSK-3b with rAxin may require the structure of GSK-3b,

which is catalytically active, and that K85 and Y216 are neces- plakoglobin and b-catenin interact with overlapping, andperhaps identical, sites on Axin. rAxin enhances the phos-sary for achieving the functional rAxin-interacting domain

of GSK-3b. phorylation of plakoglobin by GSK-3b and degrades pla-koglobin [90]. Therefore, it is likely that Axin regulates thestability of both b-catenin and plakoglobin.

b-catenin/Plakoglobin

b-catenin has been originally identified as a protein that in-Adenomatous polyposis coli gene product (APC)teracts with the cytoplasmic domain of cadherin and links

cadherin to a-catenin, which in turn mediates the anchor- APC is a tumour suppressor linked to familial adenomatouspolyposis coli (FAP) and to the initiation of sporadic hu-age of the cadherin complex to the cortical actin cytoskele-

ton [25]. Many binding partners of b-catenin have been man colorectal cancer [31]. APC encodes a 300-kDa multi-functional protein with several structural domains. Thefound, suggesting that b-catenin has other functions in ad-

dition to its role in cell–cell adhesion. Genetic and embryo- N-terminus contains an oligomerization domain followedby seven repeats of an Armadillo motif. The middle portionlogical studies have revealed that b-catenin is a component

of the Wnt signalling pathway and that it exhibits signal- of APC contains three successive 15-amino-acid (15-aa) re-peats followed by seven related but distinct 20-aa repeats,ling functions [2–4]. The primary structure of b-catenin

consists of an N-terminal portion of approximately 130 both of which repeat regions are able to bind independentlyto b-catenin [32–34]. Following the 20-aa repeats is a basicamino acids, a central region of 550 amino acids that con-

tains 12 repeats of 42 amino acids (42-aa) known as Arma- region, while the C-terminus has an S/TXV motif that in-teracts with the PDZ domain-containing human Dlg homo-dillo repeats, and a C-terminal region of 110 amino acids

[26]. rAxin interacts directly with the region containing logue [35]. In FAP and colorectal cancers, most patientscarry APC mutations, which result in the expression ofArmadillo repeats 2 to 7 of b-catenin. b-catenin binds to

amino acids 437–506 of rAxin [10] (Fig. 3). This region is truncated proteins [31]. Almost all mutant proteins lack theC-terminal-half region, including most of the 20-aa repeats,separate from and adjacent to the GSK-3b-binding site of

rAxin. GSK-3b and b-catenin bind simultaneously to but retain the 15-aa repeats. Colorectal carcinoma cellswith mutant APC contain a large amount of monomericAxin, forming a ternary complex [10]. As described below,

this complex formation enhances the phosphorylation of b-catenin [36]. Indeed, APC downregulates the level of cy-

Axin in the Wnt Signalling Pathway 781

toplasmic b-catenin when introduced into the SW480 co- domains. Both regions of Dvl-1 bind to the region con-taining amino acids 508–732 of rAxin, and they competelorectal cancer cell line [36]. This APC activity is localizedwith each other for binding to rAxin (Fig. 3). Althoughto the central region of the protein, and at least three 20-Axin also has a DIX domain in its C-terminal region, thisaa repeats are necessary for the degradation of b-cateninDIX domain does not bind directly to Dvl-1. Dvl inhibits[31, 37]. However, the role of APC in the downregulationthe ability of rAxin to enhance GSK-3b-dependent phos-of b-catenin is not clear.phorylation of b-catenin, as described below.The RGS domains of Axin and conductin interact di-

rectly with the region containing the third to seventh 20-aa repeats of APC [12, 38, 39] (Fig. 3). In this region, it is Protein Phosphatase 2A (PP2A)likely that a SAMP (Ser-Ala-Met-Pro) sequence is impor-

PP2A is one of the four major serine/threonine proteintant, since mutation of SAMP to AALP (Ala-Ala-Leu-Pro)phosphatases [50]. The catalytic subunit of PP2A (PP2Ac)in APC abolishes its binding to conductin [12]. APC isis always associated with a regulatory subunit of 65 kDatruncated at amino acids 1337 and 1427 in SW480 and(PR65 or A subunit). To this dimeric core, various third orDLD-1 cells, respectively, which are derived from humanvariable regulatory subunits (B subunits) can bind and mod-

colon cancers. The mutant APC proteins in these cells do ulate the enzymatic activity of PP2Ac [51]. Axin forms anot bind to Axin [38]. These results suggest that the inter- complex with PP2A [18], and PP2Ac binds directly to theaction of APC with Axin is important for regulating the regions containing amino acids 298-506 or 508-832 ofstability of b-catenin. rAxin, but the A subunit of PP2A alone does not [89] (Fig.

3). Whether or not the B subunit regulates the interactionof PP2Ac with Axin, and whether Axin itself is anotherDvlregulatory protein for PP2A is not known. PP2A directly

Dishevelled encodes a cytoplasmic protein of unknown bio- interacts with casein kinase 2 and CaM kinase IV, sug-chemical function in flies [40, 41]. Dishevelled mediates gesting that the phosphorylation of substrates is regulatedWg signalling during embryogenesis and adult fly develop- efficiently in the complex [52, 53]. Therefore, the physio-ment, which in turn determines the ultimate cell polarity logical significance of the complex formation betweenand fate in Drosophila [40, 41]. Genetic evidence shows that PP2A and Axin may be that the phosphorylation inducedDishevelled acts upstream of shaggy, a GSK-3b homologue, by GSK-3b is negatively regulated efficiently. Indeed,and antagonizes its functions [2–4]. Overexpression of Di- PP2A bound to Axin dephosphorylates APC and Axinshevelled in the Drosophila imaginal disc cell line clone 8 phosphorylated by GSK-3b in the Axin complex [89]. Thecauses the accumulation of Armadillo, a b-catenin homo- B subunit of PP2A interacts with APC, and expression oflogue [42, 43]. Dishevelled homologues are conserved in the B subunit reduces the level of b-catenin and inhibitsXenopus and mammals [16, 44–46]. Overexpression of Xen- the transcription of the b-catenin target gene [54]. Theseopus Dishevelled (Xdsh) induces a secondary body axis in results suggest that the B subunit may enhance the phos-Xenopus embryos [44]. In mammals, Dvl-1, -2, and -3 genes phorylation of b-catenin by inhibiting the catalytic activityhave been isolated [16, 45, 46]. Dvl-1-deficient mice are vi- of PP2Ac. Taken together, these findings suggest thatable, fertile, and structurally normal, but exhibit abnormal PP2A is present in the Axin complex and may regulate thesensorimotor gating and reduced social interaction [47]. phosphorylation of the substrates of GSK-3b.These observations suggest the redundancy of functionamong the Dvl genes in the Wnt signalling pathway and the Axin Itselfparticipation of Wnt signalling components through Dvls

Axin forms a homo-oligomer [17, 18]. The N-terminal re-in complex behavioural phenomena. All Dsh and Dvl fam-gion of rAxin, including the RGS domain, is not necessaryily members contain three highly conserved domains: anfor its self-association, while the remaining region, con-N-terminal DIX domain; a central PDZ domain, which hastaining the GSK-3b and b-catenin binding sites and thebeen shown to be a protein–protein interaction surface; andDIX domain, is necessary, but none of these individual sitesa DEP domain, which can also be found in several otheris sufficient for self-association [17]. Gel filtration columnproteins [2, 3]. Disruption of the PDZ domain abolishes itschromatography analysis of recombinant rAxin reveals thatactivity in the Wg-Armadillo pathway and in the XenopusrAxin forms a trimer or tetramer. Although the physiologi-axis induction assay [42–44]. It has been reported that thecal significance of the self-association of Axin is not known,DEP domain is critical for rescue of the dsh planar polarityit may enhance the interactions between Axin and its bind-defect and for the activation of the Jun-N-terminal kinaseing proteins and be necessary for the functions of Axin de-[48, 49].scribed in the next section.The DIX domain of Dvl-1 interacts directly with the DIX

domain of Dvl-3 [17]. Dvl-1 forms a homo-oligomer via itsFUNCTIONS OF AXINDIX domain. These results indicate that Dvls form homo-Enhancement of Phosphorylation of b-catenin and APCor hetero-oligomers. Furthermore, Dvl-1 binds to Axin [17].by GSK-3bDvl-1 has two binding regions for Axin, one in the N-ter-

minal region that contains the DIX domain, and the other b-catenin has a consensus sequence of phosphorylation sitefor GSK-3b, and b-catenin mutants lacking this site arein the C-terminal region that contains the PDZ and DEP

782 A. Kikuchi

more stable than wild-type b-catenin [55, 56]. Therefore, it oligomerization may be important for the Axin action. Al-ternatively, other proteins that are required for the degrada-is thought that the phosphorylation of b-catenin by GSK-

3b regulates the stability of b-catenin. However, it has been tion of b-catenin may bind to the DIX domain of Axin.b-catenin is a target for the ubiquitin-proteasome path-reported that GSK-3b does not significantly phosphorylate

b-catenin in vitro [22]. Axin greatly enhances the phosphor- way and the phosphorylation by GSK-3b is required for itsubiquitination [58]. In general, degradation of proteins byylation of b-catenin by GSK-3b under conditions in which

these three proteins form a complex [10, 17] (Fig. 3). The the ubiquitin-proteasome pathway involves a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzymeregion (rAxin-(298–506)) containing both the GSK-3b-

and b-catenin-binding sites is necessary and sufficient for (E2), and a ubiquitin ligase (E3) [59]. E3 is the componentof the ubiquitin-conjugating system that is generallythis activity of Axin [10]. The fact that Axin does not

increase the phosphorylation of a peptide substrate by GSK- thought to be the most directly involved in substrate recog-nition. It has been shown that F-box protein Cdc4 forms3b implies that Axin does not activate GSK-3b kinase ac-

tivity. Axin may function by placing b-catenin in the vicin- complexes with Skp1 and Cdc53 in budding yeast, and thatthis multiprotein complex functions as an E3 ubiquitin li-ity of GSK-3b, thereby enhancing the phosphorylation of

b-catenin by GSK-3b. Axil also enhances the phosphoryla- gase, which catalyzes the ubiquitination of the cyclin-dependent kinase inhibitor Sic1 in combination with thetion of b-catenin by GSK-3b [11].

GSK-3b directly phosphorylates APC, and the phos- ubiquitin-conjugating enzyme Ubc3/Cdc34 [60, 61]. F-boxproteins serve as receptors for the target proteins which arephorylation of APC enhances its binding to b-catenin in

vitro [22] (Fig. 3). Axin also facilitates GSK-3b-dependent usually phosphorylated [62, 63]. In Drosophila, a mutation ofF-box protein Slimb leads to accumulation of Armadillophosphorylation of APC [39, 89]. These results suggest that

GSK-3b and its substrates, b-catenin and APC, are simulta- [64]. Consistent with these genetic findings, bTrCP/FWD1,a mammalian homologue of Slimb, associates with b-cat-neously present in the Axin complex and that their phos-

phorylation occurs efficiently in the complex. Taken to- enin in the presence of Axin and stimulates ubiquitinationand degradation of b-catenin [65]. These results suggestgether with the observations that PP2A interacts directly

with Axin, the phosphorylation states of b-catenin and that bTrCP/FWD1 directly links the phosphorylation ma-chinery to the ubiquitination apparatus. Furthermore,APC are effectively regulated in the Axin complex. There-

fore, Axin may act as a scaffold protein, and provide well- bTrCP/FWD1 binds to IkBa in a phosphorylation-depen-dent manner and it facilitates degradation of IkBa throughmediated signal transduction that is precise, fast, and regu-

lated by feedback. the ubiquitin-proteasome pathway [66, 67]. Becauseb-catenin and IkBa share a similar amino acid sequence,the DSGXXS motif, at their phosphorylation sites impor-

Regulation of Stability of b-catenin tant for ubiquitination and degradation, this motif may be aThe observations that Axin promotes the phosphorylation consensus sequence for binding to the intracellular receptorof b-catenin and that it binds to APC suggest that Axin is bTrCP/FWD1 when the serine residues in this motif areinvolved in the regulation of the stability of b-catenin. Ex- phosphorylated [68]. Because rAxin is necessary for the for-pression of Axin in SW480 or COS cells stimulates the deg- mation of stable complexes between b-catenin and bTrCP/radation of b-catenin [38, 39, 57]. Furthermore, expression FWD1, the DIX domain of rAxin may be responsible for theof Axin in SW480 cells inhibits cellular proliferation [17]. binding to b-TrCP/FWD1.Wnt-3a induces the accumulation of b-catenin in mouse fi-broblast L cells, and the Wnt-3a-dependent increase of

Genetic Analysis of D-Axin Activity in Drosophilab-catenin is inhibited in L cells stably expressing Axin [57].Wnt-3a stimulates Tcf-4 transcriptional activity through Wingless is critical for patterning and cell fate determina-

tion in embryonic segmentation of Drosophila [2]. Embryosb-catenin, and expression of Axin inhibits Wnt-3a-depen-dent Tcf-4 activity. Thus, Axin negatively regulates the that lack the maternal d-axin protein have some denticles

on the ventral cuticle [14], which is similar to the pheno-Wnt-signalling pathway by downregulating b-catenin.Removal of the RGS domain from Axin increases its type of embryos ubiquitously expressing Wingless or Arma-

dillo [69, 70]. Wingless also plays a role in organizing legability to promote degradation of b-catenin [39]. Therefore,the RGS domain may act to inhibit the ability of Axin to structures: ectopic activation of Wingless signalling induces

supernumerary outgrowth on the dorsal side of normal legsdownregulate b-catenin, and the binding of the RGS do-main to APC may relieve this negative effect. In contrast, [71, 72]. The d-axin clone also produces a supernumerary leg

from the dorsal side of the normal leg [14]. Armadillo inter-deletion of the DIX domain from rAxin abolishes its abilityto promote the degradation of b-catenin, and to inhibit cel- acts with Pangolin/DTcf, and activates expression of target

genes, including Distal-leg (DII). The levels of DII and Ar-lular proliferation [17]. rAxin without the DIX domain canenhance GSK-3b-dependent phosphorylation of b-catenin. madillo are enhanced in the d-axin clones [14]. These re-

sults indicate that Wingless signalling is activated in em-Therefore, the phosphorylation of b-catenin is not suffi-cient for its degradation. Because the region containing the bryos lacking D-Axin. In contrast to the phenotypes

observed with the d-axin mutant clones, ectopic expressionDIX domain of Axin is necessary for its oligomerization,

Axin in the Wnt Signalling Pathway 783

FIGURE 4. Mechanism by which Wnt regulates the stability of b-catenin. b-catenin is present in the Axin complex in the absence ofWnt. In this complex, b-catenin is phosphorylated, ubiquitinated, and degraded by proteasomes. Dvl antagonizes Axin activity in re-sponse to Wnt, and b-catenin is dissociated and accumulated in the cytosol. The accumulated b-catenin translocates into the nucleusand binds to and activates Tcf/Lef, resulting in expression of the target genes.

of D-Axin generates a supernumerary leg from the ventral Asashima M., manuscript in preparation). Ventral injec-tion of this Axin mutant shortens the antero-posterior axis,side of the normal leg, and DII expression is suppressed.

These results suggest that D-Axin negatively regulates but keeps the normal head structure. Ectopic neural tissuesin dorsal side are found histologically. Injection of high-Wingless signalling. Therefore, the functions of Axin are

conserved in mammals and flies. doses of rAxin-(1–437) shows hypertrophy of endodermand notochord, but reveals no ectopic axis formation.When rAxin-(298–506), which contains the GSK-3b- and

Regulation of Axis Formation by Axin in b-catenin-binding sites, is injected into the dorsal blasto-Xenopus Embryos meres, the embryos develop almost normally. Ventral injec-Axin has been originally identified as a product of the tion of the mutant induces axis duplication, with completemouse Fused gene [5]. Duplication of the embryonic axis is head structures, similar to that of the rAxin-(228–832) mu-observed in the mice whose Axin gene is mutated [6–8]. In tant. It is clear that Axin regulates the antero-posterior andcontrast, expression of Axin in Xenopus embryos shows ven- dorso-ventral axes, but the abnormalities of the dorso-ven-tralizing phenotypes, such as loss of the head [5]. When tral pattern which are induced by the Axin mutants are notrAxin-(228–832), in which the RGS domain is deleted, is correlated with their abilities to regulate the stability ofinjected into dorsal blastomeres of Xenopus embryos, the b-catenin. For example, rAxin-(228–832) downregulatesembryos develop normally. Ventral injection of this Axin b-catenin in mammalian cells, while it induces axis dupli-mutant induces the formation of ectopic dorsal axes. The cation in Xenopus embryos, which is similar to the pheno-induced axes are complete, with well-defined eyes, cement type induced by expression of b-catenin. rAxin-(1–437)glands, notochords, somites, and neural tubes. When loses the b-catenin degradation activity and acts dominant-rAxin-(1–437), which contains the RGS domain and GSK- negatively to increase accumulation of b-catenin in mam-

malian cells, but this mutant does not induce axis duplica-3b-binding site, is injected into dorsal blastomeres, the em-bryos show reduction of anterior structures, including eyes tion in Xenopus embryos. Therefore, the functions of Axin

in early development may be different from those in cellularand cement glands (Fukui A., Kishida S., Kikuchi A. and

784 A. Kikuchi

FIGURE 5. b-catenin complexes in cytosol and nucleus. Axin regulates the intracellular distribution of b-catenin between the cytosoland nucleus. b-catenin accumulated in response to Wnt translocates into the nucleus and binds to Tcf/Lef. Tcf/Lef converts a repressorto an activator.

proliferation and differentiation of cells after birth. Activi- formation in Xenopus embryos [5]. However, Dvl-1 does notaffect the phosphorylation of peptide substrates by GSK-3b,ties of Axin in addition to regulating the stability of

b-catenin remain to be identified. irrespective of the presence or absence of Axin. Therefore,it is unlikely that Dvl inhibits GSK-3b activity directly, andit is instead likely that it inhibits GSK-3b-dependent phos-

REGULATION OF AXIN ACTIVITY BY DVL phorylation in the Axin complex. Taken together, the dataInhibition of Axin’s Ability to Enhance GSK-3b- suggest that the binding of Dvl-1 to Axin may induce con-dependent Phosphorylation formational changes of the Axin complex, which result inOverexpression of Dvl-1 in CHO cells inhibits the phos- ineffective phosphorylation by GSK-3b of its substrates.phorylation of Tau, which is known to be phosphorylatedby GSK-3 [73]. This result suggests that Dvl-1 inhibits the

Stabilization of Axin by Phosphorylationactivity of GSK-3b, which is consistent with genetic evi-dence in Drosophila that Dishevelled acts negatively up- SANDSEQQS330, SDADTLSLT341, and SLTDS343 of rAxinstream of shaggy [2, 3]. Dvl-1 directly binds to rAxin-(508– are possible phosphorylation sites for GSK-3b, because the732), which is different from the binding sites for GSK-3b, levels of phosphorylation by GSK-3b of the mutants inb-catenin, and APC [17]. The binding of Dvl-1 to Axin which serine or threonine is substituted by alanine are re-does not affect the interaction of GSK-3b, b-catenin, and duced [10]. The phosphorylation of Axin does not affect its

binding to GSK-3b or b-catenin in vitro [10]. However, ex-APC with rAxin in vitro. Dvl-1 inhibits GSK-3b-dependentphosphorylation of b-catenin and APC in the presence of pression of rAxin-(S322/326/330A) in intact cells is re-

duced in comparison with that of wild-type rAxin [74].Axin [17]. Furthermore, Dvl-1 inhibits the phosphorylationof Axin by GSK-3b [74]. These inhibitory actions of Dvl-1 Treatment of the cells with LiCl, a GSK-3b inhibitor, de-

creases the levels of Axin, whereas treatment with okadaicare eliminated by deletion of the PDZ domain, suggestingthat this domain is necessary for the inhibitory activity of acid, a phosphatase inhibitor, increases them. These results

suggest that GSK-3b regulates the stability of Axin by phos-Dvl. These results suggest that Dvl modulates Axin activityby their interaction and are consistent with the observation phorylation and that the phosphorylated form of Axin is

more stable than the unphosphorylated form. Therefore,that Dvl-1 antagonizes the ability of Axin to inhibit axis

Axin in the Wnt Signalling Pathway 785

FIGURE 6. Axin as a scaffold protein. JIP1 and MP1 act as scaffold proteins of the JNK and ERK1 pathways. Axin may act as a scaffoldprotein of the Wnt signalling pathway, in that Axin forms a complex with various components in the pathway and that it specificallytransmits the signal of Wnt to b-catenin.

GSK-3b phosphorylates both Axin and b-catenin in the In mammalian cells such as COS or L cells, Axin inter-Axin complex, and these phosphorylations have the oppo- acts with GSK-3b, b-catenin, and APC in a high molecularsite effects: the phosphorylation of Axin results in its stabi- mass complex with more than 103 kDa on gel filtration col-lization, whereas that of b-catenin leads to its degradation. umn chromatography [57] (Fig. 4). b-catenin is present in

the high molecular mass complex in the absence of Wnt-3a,while addition of Wnt-3a to the cells increases b-catenin in

Dissociation of b-catenin from the Axin Complex by Wnt a lower molecular mass complex of 200–300 kDa [57]. Incells overexpressing Axin, the Wnt-3a-induced increase ofThe Wnt proteins can be classified into two groups, theb-catenin in the low molecular mass complex is not ob-Wnt-1 and Wnt-5a classes, based on assays carried out withserved [57]. These results suggest that the balance betweenmammalian cell lines and Xenopus embryos [75–77]. Thethe high and low molecular mass complexes containingWnt-1 class includes Wnt-1, Wnt-2, Wnt-3, Wnt-3a, andb-catenin is closely regulated, and that Axin plays a role inWnt-8, which have the ability to transform cells and to pro-limiting the accumulation of b-catenin in the low molecu-mote accumulation of cytoplasmic b-catenin, while thelar mass complex. Therefore, Wnt may regulate the assem-Wnt-5a class includes Wnt-4, Wnt-5a, Wnt-5b, Wnt-7b,bly of the complex consisting of Axin, APC, b-catenin, andand Wnt-11, which do not possess the transformation andGSK-3b, and induce the dissociation of b-catenin from theb-catenin accumulation activities. Expression of Dvl-1DPDZ,complex. It is possible that b-catenin free from the complexfrom which the PDZ domain is deleted, decreases the Wnt-is accumulated, binds to different partners such as Tcf/Lef,3a-dependent accumulation of b-catenin in L cells [74].and thereby transmits transcription regulatory signals. TheWnt may induce the interaction of Dvl with Axin and in-Wnt signal may act on the Axin complex through Dvl, re-hibit Axin activities, thereby decreasing the phosphoryla-sulting in the inhibition of the GSK-3b-dependent phos-tion of b-catenin and increasing its accumulation. Further-phorylation of Axin and consequently the degradation ofmore, Wnt-3a decreases the amount of intracellular Axin,Axin. Degradation of Axin due to hypophosphorylationand expression of Dvl-1DPDZ inhibits the Wnt-3a effects onmay induce the dissociation of b-catenin from the complexAxin [74]. It is possible that Wnt downregulates Axin by

inhibiting the phosphorylation by GSK-3b through Dvl. by decreasing the binding of b-catenin to Axin.

786 A. Kikuchi

MODEL OF ROLES OF AXIN IN through a specific set of kinases that activate JNK. AnotherWNT SIGNALLING adaptor, MP1, selectively associates with MEK1 (MKK)Distribution of b-catenin between Cytosol and Nucleus and ERK1 (MAPK), but not with MEK2 or ERK2, and en-

hances activation of ERK1 by MEK1. Thus, MP1 linksb-catenin dissociated from the high molecular weight Axincomplex enters in the nucleus and forms a complex with MEK1 with ERK1.Tcf to activate transcription of Wnt target genes. Several Axin may be a scaffold protein, in that it binds to severaltarget genes have been identified, including Siamois in Xen- signalling molecules to create a multi-enzyme complex. Inopus [78], Ultrabithorax in Drosophila [79], and c-myc, the Axin complex, the phosphorylation of b-catenin, APC,c-jun, fra-1, and cyclin D1 in mammals [80–82]. These and Axin is regulated by GSK-3b and PP2A. It is notablegenes have Tcf-binding sites near or in their promoters. It that Wnt signalling affects Axin through Dvl, and inhibitsis thought that Tcf may be a transcriptional repressor rather the phosphorylation of b-catenin by GSK-3b, resulting inthan an activator, because Tcf binds to proteins that can the degradation of b-catenin. In addition to Wnt, severalmediate repression. One repressor is Groucho in Drosophila growth factors, including EGF, insulin, and IGF1, inacti-[83]. The binding sites for Armadillo and Groucho on Tcf vate GSK-3b [2, 3]. However, there have been no reportsdo not overlap, but whether or not Armadillo and Groucho showing that these growth factors cause accumulation ofbind simultaneously to Tcf is not clear. It is possible that ex- b-catenin. Therefore, Axin may act as a scaffold proteinpression of Tcf-target genes is regulated by a balance be- that selectively channels the signal from Wnt to b-catenin.tween Armadillo and Groucho. Another Tcf-binding pro- The discovery and functional analyses of Axin have pro-tein is Drosophila CBP (Creb-binding protein) [84]. CBP vided new clues as to how the stability of b-catenin is regu-interacts with the high-mobility group domain of Tcf and lated and have answered several questions about the Wntacetylates a conserved lysine in the Armadillo-binding do- signalling pathway which were posed in “Introduction”.main of Tcf. This acetylation lowers the affinity of Tcf for However, many questions still remain. How does FrizzledArmadillo. It is possible that similar repressors regulate the work? Are heterotrimeric G proteins coupled with Frizzled?Tcf activity in mammals. How is the signal transduced from Frizzled to Dvl? What is

Taken together, the findings suggest that two complexes the relationship between APC and Wnt signalling? Howcontaining b-catenin, the Axin and Tcf complexes, exist in does b-catenin translocate into the nucleus? Identificationthe cytosol and nucleus, respectively (Fig. 5). Axin regu- of new molecules that interact with known proteins in thelates the stability of b-catenin in the cytosol. When Wnt signalling pathway will contribute to understandingb-catenin binds to Tcf in the nucleus, Tcf is converted from the physiological significance and complex moleculara repressor to an activator. Wnt may regulate the subcellu-

mechanism of this pathway.lar distribution of b-catenin between the cytosol and nu-cleus.

References1. Nusse R. and Varmus H. E. (1992) Cell 69, 1073–1087.

Axin as a Scaffold Protein 2. Cadigan K. M. and Nusse R. (1997) Genes Dev. 11, 3286–3305.MAP kinase pathways are activated by multiple extracellu- 3. Dale T. C. (1998) Biochem. J. 329, 209–223.

lar stimuli. There are three MAP kinase pathways leading 4. Miller J. R. and Moon R. T. (1996) Genes Dev. 10, 2527–to activation of ERK, JNK, and p38 [85, 86]. MAP kinases 2539.

5. Zeng L., Fagotto F., Zhang T., Hsu W., Vasicek T. J., Perry IIIare activated by MAP kinase kinase (MKK) and MAP ki-W. L., Lee J. J., Tilghman S. M., Gumbiner B. M. and Costan-nase kinase kinase (MKKK). Six MAPKs, seven MKKs, andtini F. (1997) Cell 90, 181–192.seven MKKKs have been identified thus far in mammalian

6. Reed S. C. (1937) Genetics 22, 1–13.cells. Distinct signalling modules activate different MAP- 7. Gluecksohn-Schoenheimer S. (1949) J. Exp. Zool. 110,kinase groups. Because these groups can be activated by 47–76.overlapping sets of extracellular stimuli, mechanisms that 8. Jacobs-Cohen R. J., Spiegelman M., Cookingham J. C. and

Bennett D. (1984) Genet. Res. 43, 43–50.allow cells to achieve signalling specificity and to respond9. Perry III W. L., Vasicek T. J., Lee J. J., Rossi J. M., Zeng L.,correctly to changes in the extracellular environment must

Zhang T., Tilghman S. M. and Costantini F. (1995) Geneticsexist. Recently it has been demonstrated that the specificity141, 321–332.

of activation and function of MAP kinase signalling mod- 10. Ikeda S., Kishida S., Yamamoto H., Murai H., Koyama S. andules is determined by scaffold proteins that create multi- Kikuchi A. (1998) EMBO J. 17, 1371–1384.

11. Yamamoto H., Kishida S., Uochi T., Ikeda S., Koyama S.,enzyme complexes (Fig. 6). Such scaffold proteins includeAsashima M. and Kikuchi A. (1998) Mol. Cell. Biol. 18,JIP1 for the JNK pathway and MP1 for the ERK1 pathway2867–2875.[87, 88]. JIP1 binds to MLK3 (MKKK), MKK7 (MKK), and

12. Behrens J., Jerchow B.-A., Wurtele M., Grimm J., AsbrandJNK1 (MAPK), and enhances activation of JNK1 by MKK7 C., Wirtz R., Kuhl M., Wedlich D. and Birchmeier W. (1998)and MLK3. JIP1 is selective and does not bind to or en- Science 280, 596–599.hance the activity of other MAPK cascade enzymes. Thus, 13. Hedgepeth C. M., Deardorff M. A. and Klein P. S. (1999)

Mech. Dev. 80, 147–151.JIP1 is likely to be a scaffold protein that channels signals

Axin in the Wnt Signalling Pathway 787

14. Hamada F., Tomoyasu Y., Takatsu Y., Nakamura M., Nagai 46. Klingensmith J., Yang Y., Axelrod J. D., Beier D. R., PerrimonS.-I., Suzyki A., Fujita F., Shibuya H., Toyoshima K., Ueno N. N. and Sussman D. J. (1996) Mech. Dev. 58, 15–26.and Akiyama T. (1999) Science 283, 1739–1742. 47. Lijam N., Paylor R., McDonald M. P., Crawley J. N., Deng

15. Druey K. M., Blumer K. J., Kang V. H. and Kehrl J. H. (1996) C. X., Herrup K., Stevens K. E., Maccaferri G., McBain C. J.,Nature 379, 742–746. Sussman D. J. and Wynshaw-Boris A. (1997) Cell 90, 895–

16. Sussman D. J., Klingensmith J., Salinas P., Adams P. S., Nusse 905.R. and Perrimon N. (1994) Dev. Biol. 166, 73–86. 48. Boutros M., Paricio N., Strutt D. I. and Mlodzik M. (1998)

17. Kishida S., Yamamoto H., Hino S., Ikeda M., Kishida M. and Cell 94, 109–118.Kikuchi A. (1999) Mol. Cell. Biol., in press. 49. Li L., Yuan H., Xie W., Mao J., Caruso A. M., McMahon A.,

18. Hsu W., Zeng L. and Costantini F. (1999) J. Biol. Chem. 274, Sussman D. J. and Wu D. (1999) J. Biol. Chem. 274, 129–134.3439–3445. 50. Cohen P. (1989) Annu. Rev. Biochem. 58, 453–508.

19. Plyte S. E., Hughes K., Nikolakaki E., Pulverer B. J. and 51. Wera S. and Hemmings B. A. (1995) Biochem. J. 311, 17–29.Woodgett J. R. (1992) Biochim. Biophys. Acta 1114, 147–162. 52. Heriche J. K., Lebrin F., Rabilloud T., Leroy D., Chambaz E.

20. Yang S.-D., Song J.-S., Hsieh Y.-T., Liu H.-W. and Chan M. and Goldberg Y. (1997) Science 276, 952–955.W.-H. (1992) J. Prot. Chem. 11, 539–546. 53. Westphal R. S., Anderson K. A., Means A. R. and Wadzinski

21. Mandelkow E.-M., Drewes G., Biernat J., Gustke N., Van Lint B. E. (1998) Science 280, 1258–1261.J., Vandenheede J. R. and Mandelkow E. (1992) FEBS Lett. 54. Seeling J. M., Miller J. R., Gil R., Moon R. T., White R. and314, 315–321. Virshup D. M. (1999) Science 283, 2089–2091.

22. Rubinfeld B., Albert I., Porfiri E., Fiol C., Munemitsu S. and 55. Munemitsu S., Albert I., Rubinfeld B. and Polakis P. (1996)Polakis P. (1996) Science 272, 1023–1026. Mol. Cell. Biol. 16, 4088–4094.

23. Woodgett J. R. (1990) EMBO J. 9, 2431–2438. 56. Yost C., Torres M., Miller J. R., Huang E., Kimelman D. and24. Ruel L., Bourouis M., Heitzler P., Pantesco V. and Simpson P. Moon R. T. (1996) Genes Dev. 10, 1443–1454.

(1993) Nature 362, 557–560. 57. Kishida M., Koyama S., Kishida S., Matsubara K., Nakashima25. Takeichi M. (1991) Science 251, 1451–1455. S., Higano K., Takada R., Takada S. and Kikuchi A. (1999)26. Huber A. H., Nelson W. J. and Weis W. I. (1997) Cell 90, Oncogene 18, 979–985.

871–882. 58. Aberle H., Bauer A., Stappert J., Kispert A. and Kemler R.27. Mathur M., Goodwin L. and Cowin P. (1994) J. Biol. Chem. (1997) EMBO J. 16, 3797–3804.

269, 14075–14080. 59. Ciechanover A. (1994) Cell 79, 13–21.28. Troyanovsky S. M., Troyanovsky R. B., Eshkind L. G., Leube 60. Skowyra D., Craig K. L., Tyers M., Elledge S. J. and Harper

R. E. and Franke W. W. (1994) Proc. Natl. Acad. Sci. USA J. W. (1997) Cell 91, 209–219.91, 10790–10794. 61. Feldman R. M., Correll C. C., Kaplan K. B. and Deshaies R. J.

29. Ben-Ze’ev A. and Geiger B. (1998) Curr. Opin. Cell Biol. 10, (1997) Cell 91, 221–230.629–639. 62. Skowyra D., Craig K. L., Tyers M., Elledge S. J. and Harper

30. Kemler R. (1993) Trends Genet. 9, 317–321. J. W. (1997) Cell 91, 209–219.31. Polakis P. (1997) Biochim. Biophys. Acta 1332, F127–F147. 63. Feldman R. M., Correll C. C., Kaplan K. B. and Deshaies R. J.32. Rubinfeld B., Souza B., Albert I., Muller O., Chamberlain (1997) Cell 91, 221–230.

S. H., Masiarz F. R., Munemitsu S. and Polakis P. (1993) Sci- 64. Jiang J. and Struhl G. (1998) Nature 391, 493–496.ence 262, 1731–1734. 65. Kitagawa M., Hatakeyama S., Shirane M., Matsumoto M.,33. Su L. K., Vogelstein B. and Kinzler K. W. (1993) Science 262, Ishida N., Hattori K., Nakamichi I., Kikuchi A., Nakayama1734–1737. K.-I. and Nakayama K. (1999) EMBO J. 18, 2401–2410.34. Rubinfeld B., Souza B., Albert I., Munemitsu S. and Polakis

66. Yaron A., Hatzubai A., Davis M., Lavon I., Amit S., ManningP. (1995) J. Biol. Chem. 270, 5549–5555.A. M., Andersen J. S., Mann M., Mercurio F. and Ben-Neriah35. Matsumine A., Ogai A., Senda T., Okumura N., Satoh K.,Y. (1998) Nature 396, 590–594.Baeg G. H., Kawahara T., Kobayashi S., Okada M., Toyos-

67. Hatakeyama S., Kitagawa M., Nakayama K., Shirane M., Mat-hima K. and Akiyama T. (1996) Science 272, 1020–1023.sumoto M., Hattori K., Higashi H., Nakano H., Okumura K.,36. Munemitsu S., Albert I., Souza B., Rubinfeld B. and PolakisOnoe K., Good R. A. and Nakayama K.-I. (1999) Proc. Natl.P. (1995) Proc. Natl. Acad. Sci. USA 92, 3046–3050.Acad. Sci. USA 96, 3859–3863.37. Rubinfeld B., Albert I., Porfiri E., Munemitsu S. and Polakis

68. Maniatis T. (1999) Genes & Dev. 13, 505–510.P. (1997) Cancer Res. 57, 4624–4630.69. Siegfried E., Wilder E. L. and Perrimon N. (1994) Nature 367,38. Kishida S., Yamamoto H., Ikeda S., Kishida M., Sakamoto I.,

76–80.Koyama S. and Kikuchi A. (1998) J. Biol. Chem. 273, 10823–70. Zecca M., Basler K. and Struhl G. (1996) Cell 87, 833–844.10826.71. Irvine K. D. and Vogt T. F. (1997) Curr. Opin. Cell Biol. 9,39. Hart M. J., de los Santos R., Albert I. N., Rubinfeld B. and

867–876.Polakis P. (1998) Curr. Biol. 8, 573–581.72. Brook W. J. and Cohen S. M. (1996) Science 273, 1373–1377.40. Klingensmith J., Nusse R. and Perrimon N. (1994) Genes Dev.73. Wagner U., Brownless J., Irving N. G., Lucas F. R., Salinas8, 118–130.

P. C. and Miller C. C. J. (1997) FEBS Lett. 411, 369–372.41. Theisen H., Purcell J., Bennett M., Kansagara D., Syed A. and74. Yamamoto H., Kishida S., Kishida M., Ikeda S., Takada S. andMarsh J. (1994) Development 120, 347–360.

Kikuchi A. (1999) J. Biol. Chem. 274, 10681–10684.42. Yanagawa S., van Leeuwen F., Wodarz A., Klingensmith J.75. Wong G. T., Gavin B. J. and McMahon A. P. (1994) Mol.and Nusse R. (1995) Genes Dev. 9, 1087–1097.

Cell. Biol. 14, 6278–6286.43. Yanagawa S., Lee J., Haruna T., Oda H., Uemura T., Takeichi76. Du S. J., Purcell S. M., Christian J. L., McGrew L. L. andM. and Ishimoto A. (1997) J. Biol. Chem. 272, 25243–25251.

Moon R. T. (1995) Mol. Cell. Biol. 15, 2625–2634.44. Sokol S. Y. (1996) Curr. Biol. 6, 1456–1467.77. Shimizu H., Julius M. A., Giarre M., Zheng Z., Brown A. M.45. Pizzuti A., Amati F., Calabrese G., Mari A., Colosimo A., Si-

and Kitajewski J. (1997) Cell Growth Differ. 8, 1349–1358.lani V., Giardino L., Ratti A., Penso D., Calza L., Palka G.,78. Brannon M. and Kimelman D. (1996) Dev. Biol. 180, 344–Scarlato G., Novelli G. and Dallapiccola B. (1996) Hum.

Mol. Genet. 5, 953–958. 347.

788 A. Kikuchi

79. Riese J., Yu X., Munnerlyn A., Eresh S., Hsu S. C., Grosschedl 84. Waltzer L. and Bienz M. (1998) Nature 395, 521–525.R. and Bienz M. (1997) Cell 88, 777–787. 85. Treisman R. (1996) Curr. Opin. Cell Biol. 8, 205–215.

80. He T., Sparks A. B., Rago C., Hermeking H., Zawel L., da 86. Kyriakis J. M. and Avruch J. (1996) Bioessays 18, 567–Costa L. T., Morin P. J., Vogelstein B. and Kinzler K. W. 577.(1998) Science 281, 1509–1512. 87. Schaeffer H. J., Catling A. D., Eblen S. T., Collier L. S.,

81. Mann B., Gelos M., Siedow A., Hanski M. L., Gratchev A., Krauss A. and Weber M. J. (1998) Science 281, 1668–1671.Ilyas M., Bodmer W. F., Moyer M. P., Riecken E. O., Buhr 88. Whitmarsh A. J., Cavanagh J., Tournier C., Yasuda J. andH. J. and Hanski C. (1999) Proc. Natl. Acad. Sci. USA 96, Davis R. J. (1998) Science 281, 1671–1674.1603–1608. 89. Ikeda S., Kishida M., Matsuura Y., Usui H. and Kikuchi A.

82. Tetsu O. and McCormick F. (1999) Nature 398, 422–426. (2000) Oncogene in press.83. Cavallo R. A., Cox R. T., Moline M. M., Roose J., Polevoy 90. Kodama S., Ikeda S., Asahara T., Kishida M. and Kikuchi A.

G. A., Clevers H., Peifer M. and Bejsovec A. (1998) Nature (1999) J. Biol. Chem. 274, 27682–27688.395, 604–608.