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Biochemical and Biophysical Research Communications 268, 243–248 (2000)

doi:10.1006/bbrc.1999.1860, available online at http://www.idealibrary.com on

egulation of b-Catenin Signaling in the Wnt Pathway

kira Kikuchi1

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

eceived November 11, 1999

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b-Catenin not only regulates cell to cell adhesion asprotein interacting with cadherin, but also functionss a component of the Wnt signaling pathway. The Wntignaling pathway is conserved in various organismsrom worms to mammals, and plays important roles inevelopment, cellular proliferation, and differentia-ion. Wnt stabilizes cytoplasmic b-catenin and then-catenin is translocated into the nucleus where ittimulates the expression of genes including c-myc,-jun, fra-1, and cyclin D1. The amounts and functionsf b-catenin are regulated in both the cytoplasm anducleus. Its molecular mechanisms are becoming in-reasingly well understood. © 2000 Academic Press

. Wnt SIGNALING PATHWAY

Wnt proteins constitute a large family of cysteine-ich secreted ligands that control development in or-anisms ranging from nematode worms to mammals1). The outlines of the Wnt signal transduction path-ay were first elucidated by a genetic analysis of Wing-

ess signaling during the development of segmentalolarity in Drosophila, and extended through studiesf embryonic axis formation in Xenopus. In verte-rates, a number of components of the Wingless path-ay are conserved and the Wnt signaling pathway

egulates organ development and cellular prolifera-ion, morphology, motility, and fate (2–4). In the cur-ent model, the serine/threonine kinase glycogenynthase kinase-3b (GSK-3b) targets cytoplasmic-catenin for degradation in the absence of Wnt (Fig.). As a result, cytoplasmic b-catenin levels are low.hen Wnt acts on its cell-surface receptor Frizzled,vl, a cytoplasmic protein with unknown functions, isctivated. Dvl antagonizes the action of GSK-3b, per-aps by modulating its enzymatic activity. The phos-

1 Fax: 181-82-257-5134. E-mail: akikuchi@mcai.med.hiroshima-.ac.jp.

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onger degraded, resulting in its accumulation in cyto-lasm. Accumulated b-catenin is translocated into theucleus where it binds to Tcf/Lef, a transcription fac-or, and stimulates gene expression. In the nucleuseveral proteins that bind to b-catenin and Tcf/Lefegulate the complex formation of b-catenin-Tcf-DNA.herefore, it appears that b-catenin signaling is regu-

ated in both the cytoplasm and nucleus. In this review,egulation of b-catenin signaling is described.

. REGULATION OF b-CATENIN SIGNALINGY AXIN IN THE CYTOPLASM

.1. Identification of Axin

Axin was originally identified as the product of theouse gene Fused (5). An intriguing feature of many

omozygous axin mutant embryos is a duplication ofhe embryonic axis (6, 7), suggesting that Axin nor-ally plays a negative regulatory role in the response

o an axis-inducing signal. Indeed, when Axin is in-ected into Xenopus embryos, most embryos developith strong axial defects (5). Several experiments in-icate that Axin exerts its function on axis formationy specifically inhibiting the Wnt signaling (5).Independent biochemical approaches have demon-

trated the existence of Axin in various species. Axinnd its homolog, Axil (Axin like), have been identifieds proteins that bind to GSK-3b (8, 9). Another Axinomolog, conductin, has been identified as a b-catenin-inding protein (10). Conductin and Axil are the samerotein, implying that there are at least two Axin fam-ly members in mammals. The Axin gene is conservedn humans, rats, mice, chickens, and Xenopus (5, 8, 11).uman Axin and Axil are located in 16q13-3 and7q23-q24, respectively (12). When the amino acidumbers of Axin are indicated in this review, they refero rat Axin (rAxin). A homolog of the vertebrate Axinas been also found in Drosophila, and named D-Axin13, 14). Genetic evidence demonstrates that Wingless

0006-291X/00 $35.00Copyright © 2000 by Academic PressAll rights of reproduction in any form reserved.

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ignaling is activated in embryos lacking D-Axin andhat the ectopic expression of D-Axin suppressed Wing-ess signaling. Therefore, the functions of Axin areonserved in mammals and flies.

.2. Complex Formation of GSK-3b and b-Cateninwith Axin

The central region of Axin contains the GSK-3b- and-catenin-binding sites, and residues 353-437 and 437-06 of rAxin are responsible for the binding of GSK-3bnd b-catenin, respectively (8) (Fig. 2). GSK-3 wasriginally characterized as a serine/threonine kinasehat phosphorylates and inactivates glycogen synthasend is now implicated in the regulation of several phys-ological responses in mammalian cells by phosphory-ating many substrates (15). The cDNAs of mammalianSK-3a and GSK-3b have been isolated (16). Althoughoth GSK-3a and GSK-3b form complexes with Axin8), GSK-3b is mainly used in analyses of the Wntignaling pathway, since it but not GSK-3a rescues thehenotype of the Drosophila zw3/shaggy gene product,GSK-3 homolog (17). b-catenin was originally identi-ed as a protein which interacts with the cytoplasmicomain of cadherin and links cadherin to a-catenin,

FIG. 1. Mechanism by which Wnt regulates the stability of b-can this complex, b-catenin is phosphorylated, ubiquitinated, and degrnd b-catenin is dissociated and accumulated in the cytosol. The accctivates Tcf/Lef, resulting in expression of the target genes. E1,biquitin ligase; Ub, ubiquitin.

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hich in turn mediates the anchorage of the cadherinomplex to the cortical actin cytoskeleton (18). Geneticnd embryological studies have revealed that-catenin is a component of the Wnt signaling pathwaynd that it exhibits signaling functions (2–4). Axinnteracts directly with the region containing Armadilloepeats 2 to 7 of b-catenin (8). GSK-3b and b-cateninind simultaneously to different sites of Axin, formingternary complex (8). b-catenin has a consensus se-

uence of a phosphorylation site for GSK-3b (19, 20),

in. b-Catenin is present in the Axin complex in the absence of Wnt.ed by proteasome. Dvl antagonizes Axin activity in response to Wnt,ulated b-catenin is translocated into the nucleus, and binds to andiquitin-activating enzyme; E2, ubiquitin-conjugating enzyme; E3,

FIG. 2. Structure of Axin and its binding proteins. Axin pos-esses binding sites for APC, GSK-3b, b-catenin, Dvl, and PP2A. Inhe Axin complex, GSK-3b efficiently phosphorylates b-catenin,PC, and Axin itself, while Dvl and PP2A prevent the phosphoryla-

ion.

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-catenin by GSK-3b under conditions in which thesehree proteins form a complex (8, 21). Axil also en-ances the phosphorylation of b-catenin by GSK-3b (9).urther, Axin and Axil are substrates of GSK-3b andhe phosphorylation sites are close to the GSK-3b-inding region. It has been shown that phosphoryla-ion of Axin regulates its stability and its affinities for-catenin and GSK-3b (22–24).b-Catenin is a target for the ubiquitin-proteasome

athway and the phosphorylation by GSK-3b is re-uired for the ubiquitination (25). In general, degrada-ion of proteins by the ubiquitin-proteasome pathwaynvolves a ubiquitin-activation enzyme (E1), abiquitin-conjugating enzyme (E2), and a ubiquitin li-ase (E3) (26). An F-box protein is a component of E3nd serves as a receptor for the target proteins whichre usually phosphorylated (27, 28). In Drosophila, autation of F-box protein Slimb leads to accumulation

f Armadillo, a b-catenin homolog (29). Consistent withhese genetic findings, bTrCP/FWD1, a mammalianomolog of Slimb, associates with b-catenin in theresence of Axin and stimulates ubiquitination (30–2). Thus, the phosphorylation of b-catenin by GSK-3bnd its ubiquitination are enhanced in the Axin com-lex.The observations that Axin promotes the phosphor-

lation and ubiquitination of b-catenin suggest thatxin regulates the stability of b-catenin. Indeed, ex-ression of Axin and Axil (conductin) in SW480 or COSells stimulates the degradation of b-catenin (10, 33,4). Wnt-3a induces the accumulation of b-catenin inouse fibroblast L cells, and the Wnt-3a-dependent

ncrease of b-catenin is inhibited in L cells stably ex-ressing Axin (35). Wnt-3a stimulates Tcf-4 transcrip-ional activity through b-catenin, and expression ofxin inhibits Wnt-3a-dependent Tcf-4 activity (35, 36).urthermore, expression of Axin in SW480 cells inhib-

ts cellular proliferation (21). Therefore, Axin nega-ively regulates the Wnt-signaling pathway by down-egulating b-catenin. Axin also binds to plakoglobin, aomolog of b-catenin, and enhances its phosphoryla-ion by GSK-3b, resulting in the degradation of plako-lobin (37).

.3. Interaction of APC with Axin

APC is a tumor suppressor linked to familial adeno-atous polyposis coli and to the initiation of sporadicuman colorectal cancer (38). APC encodes a 300-kDaultifunctional protein with several structural do-ains. The middle portion of APC contains three suc-

essive 15-amino-acid (15-aa) repeats followed byeven related but distinct 20-aa repeats, both of whichre able to bind independently to b-catenin (39–41).he APC activity to downregulate the level of cytoplas-ic b-catenin is localized to the central region of the

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or the degradation of b-catenin (38, 42). Axin and AxilConductin) have the RGS domains in their N-termini5, 8–10). The RGS domains of Axin and conductinnteract directly with the region containing the third toeventh 20-aa repeats of APC (10, 33, 34) (Fig. 2). Mostf the APC mutants observed in human colon cancerells, where cytoplasmic b-catenin is accumulated,ave lost the region containing 20-aa repeats and doot bind to Axin (34). Therefore, the interaction of APCith Axin may be important for the APC activity toownregulate b-catenin. GSK-3b phosphorylates APCirectly (43), and the binding of APC to Axin enhancesSK-3b-dependent phosphorylation (33, 44). Although

he significance of the phosphorylation of APC is notnown, the phosphorylation of APC appears to be im-ortant for the degradation of b-catenin (42, 43). Thus,SK-3b and its substrates, b-catenin, APC, and Axin

tself, are simultaneously present in the Axin complexnd their phosphorylation occurs efficiently in the com-lex. The protein stability and functions of b-catenin,PC, and Axin are regulated in the complex.

.4. Regulation of Phosphorylation by Dvl and PP2Ain the Axin Complex

Dvl is a mammalian homolog of fly dishevelled (dsh)nd acts negatively upstream of shaggy (2, 3, 45, 46).vl-1, -2, and -3 genes have been isolated (47–49). Allsh and Dvl family members contain three highly con-

erved domains: an N-terminal DIX domain; a centralDZ domain, and a DEP domain (2, 3). The PDZ do-ain is essential for the roles of Dvl in the Wnt/ingless signaling pathway (50–52). The DEP domain

s critical for rescue of the dsh planar polarity defectnd for the activation of the Jun-N-terminal kinase53, 54). Dvl binds to Axin (21, 55–57) and inhibitsSK-3b-dependent phosphorylation of b-catenin andPC in the presence of Axin (21). Furthermore, Dvl

nhibits the phosphorylation of Axin by GSK-3b (22).hese results suggest that Dvl modulates Axin activityy their interaction and are consistent with the obser-ation that Dvl antagonizes the ability of Axin to in-ibit axis formation in Xenopus embryos (5). The bind-

ng of Dvl to Axin may induce conformational changesf the Axin complex, which result in ineffective phos-horylation by GSK-3b of its substrates.Protein phosphatase 2A (PP2A) is one of the fourajor serine/threonine protein phosphatases (58). The

atalytic subunit of PP2A (PP2Ac) is always associatedith a regulatory subunit of 65 kDa (PR65 or A sub-nit). To this dimeric core, various third or variableegulatory subunits (B subunits) bind and modulatehe enzymatic activity of PP2Ac (59). Axin forms aomplex with PP2A, and PP2Ac binds directly to theegion containing amino acids 298-506 or 508-832 ofAxin, but the A subunit of PP2A alone does not (44,

60) (Fig. 2). PP2A bound to Axin dephosphorylatesAcAobtbTmG

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PC and Axin phosphorylated by GSK-3b in the Axinomplex (44). The B0 subunit of PP2A interacts withPC, and expression of the B0 subunit reduces the levelf b-catenin and inhibits the transcription of the-catenin target gene (61). These results suggest thathe B0 subunit enhances the phosphorylation of-catenin by inhibiting the catalytic activity of PP2Ac.herefore, PP2A is present in the Axin complex anday regulate the phosphorylation of the substrates ofSK-3b.

.5. Axin as a Scaffold Protein

Axin may be a scaffold protein, in that it binds toeveral signaling molecules to create a multi-enzymeomplex (Fig. 1). In mammalian cells such as COS or Lells, Axin interacts with GSK-3b, b-catenin, and APCn a high molecular mass complex of more than 103

Da on gel filtration column chromatography (35).-catenin is present in the high molecular mass com-lex in the absence of Wnt-3a, while addition of Wnt-3ao the cells increases b-catenin in a lower molecularass complex of 200-300 kDa (35). In cells overex-

ressing Axin, the Wnt-3a-induced increase of-catenin in the low molecular mass complex is notbserved (35). These results suggest that the balanceetween the high and low molecular mass complexesontaining b-catenin is tightly regulated, and thatxin plays a role in limiting the accumulation of-catenin in the low molecular mass complex. There-ore, Wnt may regulate the assembly of the complexonsisting of Axin, APC, b-catenin, and GSK-3b, andnduce the dissociation of b-catenin from the complex.t is possible that b-catenin free from the complex isccumulated, binds to different partners such as Tcf/ef, and thereby transmits transcription regulatoryignals.Three possible mechanisms by which b-catenin is

issociated from the complex have been proposed. Therst one is that Wnt-dependent dephosphorylation ofxin induces the degradation of Axin (22). Degradationf Axin due to hypophosphorylation may induce theissociation of b-catenin from the complex. The secondne is that Wnt-induced dephosphorylation of Axinecreases its affinities for b-catenin and GSK-3b (23,4). The third possibility is that Frat1 is involved in thent signaling (57). Frat1 was originally cloned for its

umor promoting activity in lymphocytes (62), and is aomolog of Xenopus GBP, which binds to GSK-3b and

nhibits GSK-3b-dependent phosphorylation (63). Dvlnteracts with Axin and Frat1. Wnt-1 promotes theisintegration of the Frat1/Dvl/GSK-3b/Axin complex,esulting in the dissociation of GSK-3b from Axin andn the stabilization of b-catenin. In any case, b-catenins dissociated from the Axin complex in response to

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electively channel the signal from Wnt to b-catenin.

. REGULATION OF b-CATENIN SIGNALINGN THE NUCLEUS

.1. Tcf-Binding Proteins

b-catenin dissociated from the high moleculareight Axin complex enters the nucleus and forms a

omplex with Tcf to activate transcription of Wnt tar-et genes (Fig. 3). Several target genes have been iden-ified, including siamois in Xenopus (64), Ubx in Dro-ophila (65), and c-myc, c-jun, fra-1, and cyclin D1 inammals (66–68). These genes have Tcf-binding sites

ear or in their promoters. It is thought that Tcf maye a transcriptional repressor rather than an activator,ecause it binds to proteins that can mediate repres-ion. One repressor is Groucho in Drosophila (69). Theinding sites for Armadillo and Groucho on Tcf do notverlap, but whether or not Armadillo and Grouchoind simultaneously to Tcf is not clear. It is possiblehat expression of Tcf-target genes is regulated by aalance between Armadillo and Groucho. Another Tcf-inding protein is Drosophila CBP (70). CBP interactsith the high-mobility group domain of Tcf and acety-

ates a conserved lysine in the Armadillo-binding do-ain of Tcf. This acetylation lowers the affinity of Tcf

or Armadillo. It is possible that similar repressorsegulate the Tcf activity in mammals. It has beenhown that NEMO-like kinase (NLK) binds directly tond phosphorylates Tcf and that the phosphorylationf Tcf inhibits the binding of the b-catenin/Tcf complexo DNA (71). Therefore, these Tcf-binding proteins neg-tively regulate the b-catenin signaling.

.2. b-Catenin-Binding Proteins in the Nucleus

Pontin52 is a nuclear protein which binds to-catenin (72). Pontin52 can be coimmunoprecipitatedithin a large complex containing b-catenin and Lef-1nd is proposed to provide a bridging function betweenef-1/b-catenin complexes and transcriptional machin-ry (72). However, whether Pontin52 affects the-catenin signaling remains to be clarified. Duplin is aew b-catenin-binding protein found in the nucleus2

Fig. 3). Duplin does not affect the stability or subcel-ular localization of b-catenin, but competes with Tcf-4or the binding to b-catenin. Consistent with theseharacteristics, Duplin inhibits Wnt-3a- and b-catenin-ependent Tcf-4 activation in L cells and suppressesnt- and b-catenin-induced axis formation in Xenopus

mbryos. It appears that Duplin inhibits the b-cateninignaling in a manner different from Groucho, CBP,nd NLK.

2 Sakamoto, I., Kishida, S., Fukui, A., Kishida, M., Yamamoto, H.,ichiue, T., Takada, S., Asashima, M., Kikuchi, A., submitted.

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. CONCLUSION

There are multiple mechanisms to inhibit b-cateninignaling. The findings suggest that two complexesontaining b-catenin, the Axin and Tcf complexes, existn the cytoplasm and nucleus, respectively (Fig. 3).

nt may regulate the subcellular distribution of-catenin between the cytoplasm and nucleus. In theytoplasm, the amount of b-catenin is negatively reg-lated by the degradation of b-catenin in the Axinomplex. In the nucleus, gene expression induced by-catenin is negatively regulated by inhibiting theomplex formation of b-catenin, Tcf, and DNA. Muta-ions in b-catenin have been found in human cancers,ncluding colon cancer and melanoma, and the muta-ions result in the accumulation of b-catenin (73, 74).ince b-catenin functions as an oncogene, it is specu-

ated that there are several mechanisms for protectinggainst abnormal cellular proliferation by inhibiting-catenin signaling.

CKNOWLEDGMENTS

I thank my colleagues, S. Koyama, S. Kishida, M. Kishida, S.keda, H. Yamamoto, S. Hino, I. Sakamoto, S. Kodama, K. Matsu-ara, and S. Nakashima.

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FIG. 3. b-Catenin complexes in the cytoplasm and nucleus. Axinnd nucleus.

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