A Comparative Evaluation of Β-Catenin and Plakoglobin Signaling Activity

Bart O Williams*,1,3,5, Grant D Barish1,5, Michael W Klymkowsky2 and Harold E Varmus1,4

1National Cancer Institute, Division of Basic Sciences, National Institutes of Health, Bethesda, Maryland, MD 20892, USA; 2Molecular, Cellular & Developmental Biology, University of Colorado Boulder, Boulder, Colorado, CO 80309-0347, USA

Vertebrates have two Armadillo-like proteins, b-catenin about their regulation is based on studies with b- and plakoglobin. Mutant forms of b-catenin with catenin. When not bound to cadherins (Aberle et al., oncogenic activity are found in many human tumors, 1996), b-catenin is targeted for degradation via but plakoglobin mutations are not commonly found. In phosphorylation by the constitutively active glycogen fact, plakoglobin has been proposed to suppress synthase kinase 3 (GSK3) (reviewed in Dale, 1998). tumorigenesis. To assess dierences between b-catenin GSK3 forms a complex with the product of the and plakoglobin, we compared several of their biochem- tumor suppressor gene adenomatous polyposis coli ical properties. After transient transfection of 293T cells (APC) and with axin, thereby targeting b-catenin for with an expression vector encoding either of the two ubiquitin-dependent proteolysis (Behrens et al., 1998; proteins, soluble wild type b-catenin does not signi®- Farr et al., 2000; Hart et al., 1998; Ikeda et al., 1998; cantly accumulate, whereas soluble wild type plakoglobin Sakanaka et al., 1998; Yamamoto et al., 1998; Zeng is readily detected. As anticipated, b-catenin is stabilized et al., 1997), mediated by the F-box protein Slimb/ b- by the oncogenic mutation S37A; however, the analogous TrCP (Hart et al., 1999; Jiang and Struhl, 1998; mutation in plakoglobin (S28A) does not alter its half- Kitagawa et al., 1999; Latres et al., 1999; Liu et al., life. S37A-b-catenin activates a TCF/LEF-dependent 1999; Margottin et al., 1998; Marikawa and Elinson, reporter 20-fold more potently than wild type b-catenin, 1998; Winston et al., 1999). In the presence of a Wnt and *5-fold more potently than wild type or S28A signal, GSK3 activity is inhibited, and cytosolic levels plakoglobin. These dierences may be attributable to an of b-catenin rise (Cook et al., 1996; Dale, 1998). enhanced anity of S37A b-catenin for LEF1 and Cytosolic b-catenin enters the nucleus where it can TCF4, as observed here by immunoprecipitation assays. heterodimerize with members of the T-Cell Factor/ We show that the carboxyl-terminal domain is largely Lymphoid Enhancer Factor-1 (TCF/LEF) subfamily responsible for the dierence in signaling and that the of high mobility group (HMG) box DNA binding Armadillo repeats account for the remainder of the proteins (Behrens et al., 1996; Huber et al., 1996; dierence. The relatively weak signaling by plakoglobin Molenaar et al., 1996; van de Wetering et al., 1997). and the failure of the S28A mutation to enhance its Binding of b-catenin alters the activity of TCFs, stability, may explain why plakoglobin mutations are thereby aecting target gene transcription (reviewed infrequent in malignancies. Oncogene (2000) 19, 5720 ± in Barker et al., 2000). A number of transcriptional 5728. targets for Wnt signaling have been identi®ed, including the dorsalizing genes Siamois (Brannon et Keywords: b-catenin; plakoglobin; Wnt; LEF/TCF al., 1997; Carnac et al., 1996; Fan et al., 1998; Kessler, 1997), Twin (Laurent et al., 1997), Xnr3 (McKendry et al., 1997); the proto-oncogenes c-myc Introduction (He et al., 1998) and cyclin D1 (Tetsu and McCormick, 1999); ®bronectin (Gradl et al., 1999); Wnts were originally identi®ed as key regulators of and the gene encoding the matrix metalloprotease early development in Drosophila and as proto- matrilysin (Crawford et al., 1999). oncogenes in mammals (Nusse and Varmus, 1992). In addition to their interactions with TCF/LEFs, Wnts are secreted polypeptides that bind to members ALPs also serve to anchor actin ®laments to adherens of the Frizzled receptor family, triggering a series of junctions by binding simultaneously to classical cytosolic events that increase the stability of the cadherins and to the actin-binding protein a-catenin armadillo protein (Drosophila) or its homologs in (reviewed in Aberle et al., 1996). Vertebrates contain a higher vertebrates (b-catenin and plakoglobin, or second type of cadherin-based adherens junction, the Armadillo-like proteins [ALPs]), ultimately leading desmosome, which serves to anchor the intermediate to changes in gene expression (reviewed in Dale, ®lament network to the plasma membrane (Kowalczyk 1998). Subsequent studies have begun to resolve the et al., 1999). Plakoglobin is specialized to bind to molecular mechanism underlying regulation of these desmosomal cadherins (Gelderloos et al., 1997; Wahl et ALP's by Wnt signaling. Much of what is known al., 1996; Witcher et al., 1996); b-catenin normally does not bind to these proteins, but can if plakoglobin is absent, as is the case in mice engineered to lack *Correspondence: BO Williams plakoglobin (Bierkamp et al., 1999). Binding of Current addresses: 3Van Andel Research Institute 333 Bostwick NE plakoglobin to desmosomal cadherins blocks plakoglo- Grand Rapids, MI 49503, USA; 4Memorial Sloan-Kettering Cancer bin's ability to bind to a-catenin, thereby inhibiting the Center, 75 York Avenue, New York, NY 10021, USA 5These two authors contributed equally to this work interaction of actin ®laments (Gelderloos et al., 1997). Received 9 June 2000; revised 8 September 2000; accepted 13 Drosophila does not appear to have cytoplasmic September 2000 intermediate ®laments, and therefore has only a single Comparison of b-catenin and plakoglobin BO Williams et al 5721 type of cadherin-based adherens junction and a single have mutations in b-catenin (Sparks et al., 1998). In b-catenin/plakoglobin ortholog, Armadillo (Gelderloos addition, b-catenin mutations are found in *25% of et al., 1997). Both plakoglobin and b-catenin have been melanoma cell lines (Rubinfeld et al., 1997), *30% of shown to rescue the adhesion defects associated with hepatocellular carcinomas and cell lines (Huang et al., mutations in Armadillo (White et al., 1998). In 1999; Kondo et al., 1999; Legoix et al., 1999; Miyoshi contrast, neither b-catenin nor plakoglobin can com- et al., 1998; Nhieu et al., 1999; Ogawa et al., 1999), pletely rescue the defects in Wnt signaling associated *75% of pilomatricomas (Chan et al., 1999), and with Armadillo mutations in ¯ies; in fact, both mimic smaller percentages of several other tumor types the eects of dominant-negative Armadillo mutations in (reviewed in Polakis, 1999). With few exceptions, these some tissues (White et al., 1998). are point mutations in b-catenin's putative GSK3 The amino terminal domains of b-catenin and phosphorylation domain, and lead to increased plakoglobin contain a highly conserved consensus stability and accumulation of cytoplasmic and nuclear sequence for phosphorylation by GSK3 (Figure 1b), b-catenin (Polakis, 1999). In contrast, only one report which is thought to regulate their post-translational of an analogous mutation in plakoglobin has been stability (reviewed in Polakis, 1999). Phosphorylation reported in a human tumor (Caca et al., 1999). of multiple sites is believed to target ALPs for Moreover, loss of heterozygosity at the plakoglobin ubiquitin-dependent proteolytic degradation. Hsu et locus has been observed in human breast and ovarian al. (1998) also identi®ed an apparent transcriptional cancer (Aberle et al., 1995), and it has been proposed transactivator in the N-terminal region of b-catenin. that plakoglobin can act as a tumor suppressor ALPs contain a central `Armadillo repeat' region (Simcha et al., 1996). Consistent with this proposal, composed of 12 repeats of a *42 amino acids motif. reduced levels of plakoglobin RNA have been found in This region forms a positively charged groove (Huber cervical carcinoma (Denk et al., 1997) and are linked et al., 1997) and mediates heterotypic interactions, to unfavorable prognosis in patients with non-small including binding to cadherins (Aberle et al., 1996; cell lung carcinoma (Pantel et al., 1998). Barth et al., 1997), Axin/Axil (Behrens et al., 1998; To gain insight as to why b-catenin mutations are Ikeda et al., 1998; Kikuchi, 1999a,b), APC (Rubinfeld much more commonly encountered than mutations in et al., 1993; Su et al., 1993), Fascin (Tao et al., 1996), plakoglobin in human tumors, we undertook a variety and SOXs (Zorn et al., 1999). Its structural similarity of studies to compare the activities of these two ALP's to the nuclear localization sequence receptors, the in normal and mutant forms. We found dierences in karyopherins/importins, presumably explains why b- stability of the two proteins in human 293T cells, in catenin can enter the nucleus in a karyopherin/ their anities for the LEF-1 and TCF-4 DNA binding importin- and RAN-independent manner (Fagotto et proteins, and in their ability to transactivate a LEF/ al., 1998; Yokoya et al., 1999). The carboxyl terminus TCF-responsive reporter (OT). We have used chimeric of the ALPs has been identi®ed as a transactivation constructs to map the activities of dierent regions of domain and is critical for Armadillo signaling (Hsu et the polypeptides. Together these studies provide a al., 1998; Orsulic and Peifer, 1996; Simcha et al., 1998). plausible model that explains the dierences in the Numerous reports have suggested a role for b- oncogenic potentials of these two similar proteins. catenin in human tumorigenesis (reviewed in Polakis, 1999). Approximately 85% of colon carcinoma cell lines are null for APC and contain elevated levels of Results cytosolic b-catenin (Korinek et al., 1997); of the colon tumors that are wild type for APC, approximately 50% Expression of exogenous b-catenin and plakoglobin reveals differences in protein stability To compare the properties of b-catenin and plakoglobin, cDNAs for the wild type versions of both genes were cloned into the same expression vector and transfected into human 293T cells. Since Wnt signaling is believed to occur through regulation of the levels of Armadillo-related proteins by an ubiquitin-dependent, phosphorylation-sensitive process, we ®rst measured the accumulation of soluble forms of the two proteins by Western blot analysis. In conditions under which we observe abundant amounts of plakoglobin, we detected little or no increase in b-catenin over endogenous levels (Figure 2a), suggesting either an unanticipated dierence in the rates of synthesis from the two closely related expression plasmids or, more likely, a higher rate of b-catenin degradation. Figure 1 Features of b-catenin and plakoglobin. (a) b-catenin We next compared the eect of an oncogenic point and plakoglobin can be conceptually divided into three regions as mutation in b-catenin (S37A b-catenin) and an described and separated by bars. The per cent amino acid analogous mutation in plakoglobin (S28A Plakoglobin) similarity is shown between b-catenin and plakoglobin is shown (see Figure 1b) on protein levels. As previously for each region. (b) Amino acid sequences of a portion of the GSK3 regulatory domain for b-catenin and plakoglobin. The reported for similar mutants in b-catenin (Morin et serine residue underlined is the one in which an alanine al., 1997; Por®ri et al., 1997; Rubinfeld et al., 1997; substitution was made for the studies described Yost et al., 1996), immunoblot analysis of lysates

Oncogene Comparison of b-catenin and plakoglobin BO Williams et al 5722 contrast, b-catenin was readily detected in cells transfected with the S37A b-catenin expression plasmid, and the mutant exhibited a half-life of greater than six hours (Figure 2b). Wild type or S28A plakoglobin both showed intermediate stability, with a half-life of approximately two hours. Thus, we conclude that wild type b-catenin is more susceptible than wild type plakoglobin to proteolytic degradation, and mutations in the amino terminally portions have dramatically dierent eects on stability, at least in 293T cells.

Expression of exogenous b-catenin and plakoglobin reveals differences in signaling activity Excessive transcription of the genes regulated by b- catenin-TCF/LEF heteroduplexes is thought to underlie b-catenin's role in oncogenesis (reviewed in Barker et al., 2000). To compare the transcriptional activities of b-catenin and plakoglobin, constructs encoding various forms of the two proteins were transfected along with a TCF/LEF-dependent luciferase reporter construct (OT) into 293T cells. Transfection of wild type b-catenin into 293T cells had minimal eects on the activity of the OT reporter ± only threefold induction when 4 mg of plasmid were introduced (Figure 2c). In contrast, transfection of wild type plakoglobin resulted in larger increases in transactivation ± up to 17-fold when 4 mg of wild type plakoglobin expression plasmid were introduced (Figure 2c). Endogenous or exogenous expression of serine/ threonine point mutants in b-catenin is known to increase transcription at TCF/LEF target gene promoters (Morin et al., 1997; Por®ri et al., 1997; Figure 2 Expression, stability, and transactivation potential of Rubinfeld et al., 1997). Introduction of as little as wild type and mutant b-catenin and plakoglobin. (a) Western blots to determine cytoplasmic levels of b-catenin and plakoglo- 0.2 mg (data not shown) or as much as 3.0 mgof bin. Top panel measures b-catenin levels 48 h after transfection of S37A b-catenin plasmid DNA into 293T cells 293T cells with indicated amounts (in mg) of wild-type (WT) or potently transactivated the OT reporter, roughly 80- mutant b-catenin DNA (S37A). The bottom panel shows fold over baseline (Figure 2c). Although the S37A cytoplasmic plakoglobin in 293T cells transiently transfected with the indicated amount (in mg) of wild type and mutant plakoglobin mutant form of b-catenin signaled *25-fold more expression constructs (see Materials and methods). (b) Pulse/chase strongly than wild type b-catenin, S28A Plakoglobin analysis and plakoglobin proteins. Shown are the per cent of signaled no better than wild type Plakoglobin. For protein at each time point relative to the start of the chase period. either form of Plakoglobin, 15 ± 20-fold transactiva- (c) Relative fold activation of the TO reporter induced by tion was the largest observed at any dose, merely a increasing amounts of the indicated plasmids after transient transfection of 293T cells quarter of that observed for oncogenic, S37A b- catenin (Figure 2c).

derived from cells transfected with S37A b-catenin- expressing DNA displayed a large increase in the Both the carboxyl and amino termini of b-catenin are amount of soluble b-catenin (Figure 2a). In contrast, necessary for full transcriptional transactivation expression of the S28A mutant form of plakoglobin To assess the contribution of the ALP domains to the did not increase soluble plakoglobin above the overall signaling capacity of b-catenin, we tested amounts seen in cells transfected with plasmid constructs in which portions of the amino or carboxyl encoding wild type plakoglobin (Figure 2a). Moreover, terminus of b-catenin were deleted. Munemitsu et al. expression of plakoglobin, either the wild type or the (1996) created and characterized a mutant of b-catenin S28A mutant form, increased soluble endogenous b- lacking the amino terminal 89 amino acids (N89, catenin (data not shown), consistent with other reports Figure 3a), and we tested this construct in the TCF- (Miller and Moon, 1997, Simcha et al., 1998). dependent reporter gene assay. The N89 mutant b- To determine whether the dierences between the catenin transactivated the OT reporter approximately levels of soluble b-catenin and plakoglobin re¯ected 15 ± 20-fold over baseline levels ± one-®fth as active as dierences in the stabilities of the two proteins, we the full length S37A form of b-catenin (Figure 3b). The measured protein half lives by pulse-chase methods in dierence between the N-terminal deletion and point transiently transfected 293T cells (Figure 2b). We could mutant forms of b-catenin cannot be ascribed to not detect soluble b-catenin in cells transfected with a protein stability, as they accumulate to similar levels wild type b-catenin expression plasmid, even immedi- based on Western blots (Figure 3c). Thus, like Hsu et ately after the labeling period (data not shown). In al. (1998) and Caca et al. (1999), we conclude that at

Oncogene Comparison of b-catenin and plakoglobin BO Williams et al 5723 least part of the transactivation potential resides in the of ®ve times. The data shown are from a representative amino terminal domain. experiment. Constructs in which the amino terminus of Two deletions of the b-catenin carboxyl terminus S37A b-catenin replaced the amino terminus of were generated, removing the ®nal 72 (C72) or 14 plakoglobin (B-P-P) did not transactivate OT to any (C14) amino acids (Figure 3a). Transfection of either greater degree than S28A plakoglobin (Figure 4b). For carboxyl terminal deletion construct into 293T cells unclear reasons, the reciprocal construct (P-B-B) did results in mild transactivation of the OT reporter ± not accumulate to usual levels in 293T cells (data not about 10 ± 20% of that induced by similar amounts of shown). Chimeric constructs in which the Armadillo full length S37A b-catenin (Figure 3c and data not repeats of the two proteins were exchanged indicate shown). Again, dierences in transactivation could not that roughly 25% of b-catenin's enhanced signaling be ascribed to dierences in protein expression between capacity relative to that of plakoglobin localizes to the the wild type and the C14 mutants since they were Armadillo repeats (compare S37A b-catenin to B-P-B expressed at similar levels (Figure 3c). We failed to detect the C72 mutant, which we believe indicates that the epitope recognized by the b-catenin antibody used in these studies lies between 72 and 14 amino acids from the carboxyl terminus. Thus, we conclude that residues in both the amino terminal 89 and carboxyl terminal 14 residues of b-catenin are necessary either for its full transcriptional activity or its normal entry into the nucleus.

Creation of chimeric proteins to identify functional domains of b-catenin and plakoglobin To determine the relative contribution of ALP domains to the dierential signaling capacities of b-catenin and plakoglobin, we generated chimeric b-catenin/plakoglobin fusion constructs. As described above, b-catenin and plakoglobin can be conceptually divided into three portions: the amino terminal region, the Armadillo repeats, and the carboxyl terminal domain (Figure 1a). Chimeric proteins containing several combinations of domains were created from the parental S37A b- catenin and S28A plakoglobin constructs. These fusions are represented as three letter acronyms that correspond to the b-catenin (B) or plakoglobin (P) source for the amino terminus, Armadillo repeats, and carboxyl terminus, respectively (Figure 4a). At least two independently derived constructs were tested for each chimera and each construct was tested a minimum

Figure 4 Chimeric b-catenin/Plakoglobin constructs identify regions of b-catenin and plakoglobin that account for the dierential activity of the two proteins in transactivation assays. (a) Schematic representation of the chimeric constructs used in this study. The proteins are conceptually divided into three regions (as shown in Figure 1). A ®lled box denotes a portion of the chimeric protein derived from S37A b-catenin while an open box indicates that the region was derived from S28A plakoglobin. An example of the nomenclature used is as follows: P-B-B contains the amino terminus of S28A plakoglobin, the Arm repeats of b-catenin, and the carboxyl terminus of b-catenin. (b) Activity of the OT reporter in lysates of 293T cells after transient transfection with the indicated chimeric protein expression Figure 3 Deletions of either the N or C terminal portions of b- constructs. (c) Anti-b-catenin Western blot of cytoplasmic extracts catenin dramatically reduce its activity. (a) Schematic diagram of of cell lysates derived from 293T cells transiently transfected with the deletion mutants described in the text. (b) Activity of these the denoted expression plasmid. The antibody used speci®cally deletion mutants when plasmids driving their expression were recognizes the C terminus of b-catenin. (d) Anti-plakoglobin transiently transfected into 293T cells. Activity on the cotrans- Western blot of cytoplasmic extracts of cell lysates derived from fected OT reporter relative to a control transfection with empty 293T cells transiently transfected with the denoted expression vector is shown. (c) Western blot analysis of cytoplasmic and plasmid. The antibody used speci®cally recognizes the C terminus membrane-associated fractions of lysates from cells transfected of plakoglobin. Note that in both (c) and (d) the origin of the C with the indicated constructs terminal domain correlates with detection of the hybrid protein

Oncogene Comparison of b-catenin and plakoglobin BO Williams et al 5724 in Figure 4b). Constructs in which the carboxyl termini of b-catenin and plakoglobin were swapped (P-P-B and B-B-P) suggest that as much as 70% of b-catenin's relative signaling potency stems from dierences in the carboxyl termini. Western analysis of protein extracts from cells transiently transfected with the chimeras, using antibodies speci®c for the carboxyl termini of b- catenin and plakoglobin, con®rmed that each (except for B-P-P- data not shown) was well expressed (Figure 4c,d). Western analysis using antibodies to the amino terminus of plakoglobin con®rmed that the full length plakoglobin and the P-P-B chimeric constructs were equally expressed (data not shown). Importantly, we detected a small but consistent increase in the cytoplasmic levels of endogenous b- catenin with transfection of either S28A plakoglobin or chimeric constructs containing the plakoglobin C- terminus into 293T cells (Figure 4c). Such an increase in endogenous b-catenin protein levels in the cytosol is consistent with the proposal by Miller and Moon that introduction of exogenous b-catenin or plakoglobin can induce accumulation of endogenous b-catenin, presumably by overwhelming the degradative machinery (Miller and Moon, 1997).

b-catenin and plakoglobin bind differentially to regulators and effectors of Wnt signaling Figure 5 b-catenin and plakoglobin both interact well with Axin Our work suggests that, at least in 293T cells, there are but interact dierentially with LEF1 and TCF4. (a) Co- dierences in the regulation and signaling activity of b- immunoprecipitation of myc-tagged axin with V5-tagged b- catenin and plakoglobin. To gain further insight into catenin and plakoglobin. Plasmids transfected into the 293T cells these dierences we examined the relative ability of the used to make cell lysates are indicated on the right of the three panels. Left panel: Western blot of total cell lysate for myc-tagged two proteins to interact with known binding partners. axin. Middle panel: Western blot for V5 tagged constructs. Right Axin is one of two related suppressors of Wnt signaling panel: Immunoprecipation with V5 antibody followed by Western that are implicated in the formation of a complex that blotting for myc-tagged axin. (b, c) Co-immunoprecipitation of also contains GSK3, APC, catenins, dishevelled, and V5-tagged hTCF4 or mLEF1 with b-catenin or plakoglobin. (b) other proteins (Behrens et al., 1998; Fagotto et al., Anti-V5 Western blot analysis of whole cell lysates of V5 tagged hTCF4 (lanes 1 and 3) or mLEF1 (lanes 2 and 4) cotransfected 1999; Farr et al., 2000; Hart et al., 1998; Ikeda et al., with either S37A b-catenin or S28A Plakoglobin. (c) Co- 1998; Smalley et al., 1999; Yamamoto et al., 1998). immunoprecipitation from same lysates shown in (b). Immuno- This large complex is believed to regulate the precipitates formed with anti- b-catenin (lanes 1A and 2A), anti- phosphorylation, stability, and function of b-catenin. plakoglobin (lanes 3A and 4A), or an irrelevant primary antibody (Lanes 1B, 2B, 3B, 4B) were assayed by Western blot, using anti- To compare the axin binding activity of the two V5 antibody to detect Tcf4-V5 or Lef1-V5 armadillo homologues, we constructed plasmids encoding forms of both proteins tagged with the V5 epitope (see Materials and methods) and introduced each of them into 293T cells in the company of a plasmid detectable under the conditions used in this assay. encoding a myc-tagged form of mouse axin. Anti-V5 Thus, b-catenin seems to interact more strongly with immunoprecipitates from the cell lysates showed that LEF1 and TCF4 than does plakoglobin. both b-catenin and plakoglobin interacted eciently with axin (Figure 5a). Neither b-catenin nor plakoglobin contain DNA Discussion binding domains, but form heterodimers with TCF/ LEF DNA binding proteins to regulate target b-catenin mutations occur in numerous human tumors promoter activity. We reasoned that dierential (reviewed in Polakis, 1999). Plakoglobin is a gene anities for TCF/LEF may underlie the dierent highly related to b-catenin (Figure 1a). It encodes a abilities of these proteins to transactivate the OT protein containing similar consensus motifs for phos- reporter used in these studies. To this end, we phorylation by GSK3 (Figure 1b) and is postulated to compared the abilities of b-catenin and plakoglobin associate with similar proteins in the cell (Gelderloos et to interact with LEF1 and TCF4 (5b,c). As previously al., 1997), yet only one case of a plakoglobin mutation reported (reviewed in Barker et al., 2000), b-catenin in a human tumor has been reported (Caca et al., binds to both LEF1 and TCF4, although, under the 1999). To gain insight into why mutations in b-catenin, conditions used in this experiment, the interaction with but not plakoglobin, are commonly found in human TCF4 was consistently stronger. Plakoglobin also tumors, we transfected plasmids directing the expres- bound to TCF4, although the interaction appeared to sion of wild type versions of both proteins into 293T be weaker than the TCF4/b-catenin interaction. An cells. Whole cell lysates from these transfections interaction between plakoglobin and Lef1 was barely showed that transfection of a wild type plakoglobin

Oncogene Comparison of b-catenin and plakoglobin BO Williams et al 5725 expression vector results in increased plakoglobin levels al., 1999; Korinek et al., 1997; Morin et al., 1997; in the cytoplasm. In contrast, introduction of an Por®ri et al., 1997) and consistent with the increased equivalent amount of wild type b-catenin expression levels of b-catenin protein discussed previously (Figure vector was not associated with increased b-catenin 2a), transfection of cells with the S37A version of b- levels in 293T cells. This was due to a shorter half-life catenin resulted in a dramatic increase in the of the b-catenin protein as shown by pulse/chase transactivation of the reporter relative to wild type b- analysis (Figure 2c and data not shown). Thus, we catenin expression plasmid. In contrast, the wild type conclude that, at least in 293T cells, the levels of b- and S28A plakoglobin expression plasmids were similar catenin and plakoglobin proteins in the cytoplasm are in their ability to transactivate the reporter. Although dierentially regulated. Consistent with this, transient Caca et al. (1999) previously noted a small increase in transfection of a Wnt1 expression plasmid into 293T the ability of a similarly mutated (S28L) plakoglobin cells leads to an increased amount of cytoplasmic b- molecule to transactivate a LEF/TCF responsive catenin while not inducing a similar accumulation of reporter, both reports are consistent with the idea that cytoplasmic plakoglobin (data not shown). the mutation in the b-catenin protein results in more The protein levels of b-catenin, and potentially potent transactivation than a similar mutation in plakoglobin, are regulated by a multiprotein complex plakoglobin. We feel that these observations may help formed on the scaolding protein axin (Behrens et al., explain why point mutations in b-catenin are more 1998; Fagotto et al., 1999; Hart et al., 1998; Ikeda et commonly associated with tumorigenesis than similar al., 1998; Kodama et al., 1999; Sakanaka et al., 1998; mutations in plakoglobin. That is, mutations in b- Smalley et al., 1999; Yamamoto et al., 1998). To catenin cause a dramatic increase in the cytoplasmic determine if anity for axin could account for the levels of the protein and its subsequent signaling dierential protein stabilities observed, we examined activity, whereas analogous mutations in plakoglobin the ability of the two proteins to associate with axin do not confer extra activity relative to the wild type and found that both could be eciently coprecipitated version. It should be noted that in some rare situations with axin in extracts from 293T cells (Figure 5a). this might not be the case. In the BT549 breast cancer Another possibility is that interactions of b-catenin and cell line, which lacks E-cadherin expression, an plakoglobin with components of the ubiquitin-depen- analogous point mutation in plakoglobin does result dent proteolytic system, following phosphorylation by in an approximately ®vefold increase in transactivation GSK3, could account for their dierences in stability. relative to the introduction of a plasmid expressing the Following phosphorylation by GSK3, the proteins are wild type version of plakoglobin (Caca et al., 1999). targeted for degradation via interaction with the b-catenin and plakoglobin lack DNA binding vertebrate homolog (b-TrCP) of the Drosophila slimb domains and must bind to HMG box-containing, protein (Hart et al., 1999; Jiang and Struhl, 1998; DNA binding proteins in order to regulate transcrip- Kitagawa et al., 1999; Latres et al., 1999; Liu et al., tion. To assess how such interactions may contribute to 1999; Margottin et al., 1998; Marikawa and Elinson, dierences in transactivation potential, we performed 1998; Winston et al., 1999). Sadot et al. (2000) recently immunoprecipitation analysis on 293T cells cotrans- reported that both b-catenin and plakoglobin interact fected with b-catenin or plakoglobin and either of two with b-TrCP in 293 cell extracts. They further speculate members of the LEF/TCF family, LEF1 and TCF4. As that plakoglobin may have a stronger anity for the assessed by immunoprecipitation, b-catenin could cadherin-based cytoskeletal system and that this may interact with TCF4 and LEF1. Plakoglobin could also render it less susceptible to the proteolytic machinery. interact with TCF4, but at a reduced eciency We agree that dierences in protein stability are likely compared to b-catenin. Very little if any LEF1 was to be cell type-speci®c (Papko et al., 1996), depending found to co-immunoprecipitate with plakoglobin. This on the relative expression levels of a variety of is consistent with the demonstration of Simcha et al. interacting proteins including cadherins. (1998) who reported that b-catenin-containing nuclear To further compare the two proteins, a mutation in structures were more ecient at recruiting LEF1 than b-catenin found in human tumors (S37A) (Polakis, plakoglobin-containing nuclear structures. However, 1999) and the analogous mutation in plakoglobin others have reported that b-catenin and plakoglobin (S28A) were created and cloned into expression vectors were both eective in immunoprecipitating LEF1 for transfection of 293T cells. Western blots of whole (Zhurinsky et al., 2000; Huber et al., 1996). Perhaps cell lysates showed that, consistent with many previous the use of dierent buers for the immunoprecipitation reports of mutations in the GSK3 consensus sites of b- reactions could account for these dierences. catenin, the S37A point mutation resulted in a In light of the signaling discrepancy between S37A dramatic increase the cytoplasmic levels of b-catenin b-catenin and S28A plakoglobin, we attempted to protein. Consistent with another report (Caca et al., identify regions of these homologs that account for this 1999), transfection of an expression plasmid encoding dierence. Chimeric versions of the proteins were the analogous mutation in plakoglobin did not result in created by dividing them into three conceptually increased levels of plakoglobin protein in whole cell de®ned regions: the central region of the proteins lysates. Pulse/chase analysis shows that there is no containing the Armadillo repeats (Arm), and the detectable dierence in the half-lives of S28A plako- regions on the amino (NT) and carboxyl (CT) sides globin and the wild type plakoglobin protein in 293T of the armadillo repeats (Figure 1). These constructs cells (Figure 2c). were assayed for their ability to transactivate the OT To further compare the activities of the two proteins, reporter, revealing that most of the dierence between we assayed the abilities of the wild type and point the signaling activities of these homologs maps to the mutated constructs to transactivate a LEF/TCF carboxyl terminal region. Analysis of other chimeric responsive reporter (OT). As seen by others (Caca et constructs also implicated regions within the armadillo

Oncogene Comparison of b-catenin and plakoglobin BO Williams et al 5726 repeats in the dierential signaling of b-catenin and TCF4 (H Clevers), and Axin (F Constantini). The expression plakoglobin. The fact that the dierences between the vectors pCDNA3, pCDNA3.1, and pCDNA6 were purchased two proteins map most signi®cantly to the carboxyl from Invitrogen. A LEF/TCF responsive reporter (OT, an terminus is somewhat surprising based on previous optimized version of TOPFLASH) was obtained from B observations (Hecht et al., 1999; Hsu et al., 1998) that Vogelstein. The amino terminal deletion form of b-catenin was obtained from P Polakis. the carboxyl termini of both proteins strongly transactivates a heterologous reporter when fused to the relevant DNA binding domain (either Gal4 or LexA). The reason for this dierence is not clear. It is Creation of constructs used in this study notable, however, that the carboxyl terminal amino All PCR reactions were performed using Pfu polymerase acid sequence is the most divergent region between b- (Stratagene). For each construct, at least two independently catenin and plakoglobin (Figure 1a). derived clones were tested and found to be similar in their One question that arises from these studies is whether characteristics. Wild type human b-catenin sequence was plakoglobin has any intrinsic signaling activity. It has ampli®ed using primers to create a 5' Asp718 site upstream of an optimized sequence for translational initiation and a 3' been postulated that the signaling activity observed when BamHI site immediately after the stop codon. This fragment plakoglobin is overexpressed in tissue culture cells and in was cloned into the Asp718 and BamHI sites of pCDNA3. the developing Xenopus embryo is due to its ability to Wild type human plakoglobin sequence was ampli®ed using interact with elements of the b-catenin ± ubiquitin- primers to create a 5' EcoRI site upstream of an optimized dependent proteolytic degradation system and thereby Kozak sequence and ATG and a 3' XhoI site after the stop cause an elevation in the levels of endogenous b-catenin codon. The fragment was cloned into the EcoRI and XhoI (Miller and Moon, 1997). The fact that Western analysis sites of pCDNA3. S37A b-catenin and S28A plakoglobin of lysates of cells into which plakoglobin constructs have point mutants were created by site-directed mutagenesis of been transfected shows increases in cytosolic b-catenin the respective wild type constructs using a Quick-Change levels (Figure 4c) is consistent with this hypothesis. Mutagenesis Kit (Stratagene). In both cases, mutant clones were identi®ed by the presence of an NgoMI site (introduced However, the carboxyl terminal region of plakoglobin by the mutagenic oligonucleotides) using restriction digest has been shown to have intrinsic signaling activity analysis. (Simcha et al., 1998). In addition, plakoglobin, but not To create chimeric constructs with portions of b-catenin b-catenin, has been shown to transactivate a reporter and plakoglobin, plakoglobin cDNA was mutagenized to gene downstream of the myc promoter (Kolligs et al., introduce new restriction sites near the 5' (A¯3) and 3' (NdeI) 2000). Moreover, a version of plakoglobin deleted for the ends of the sequences encoding the Armadillo repeats. The amino terminal domain and ®rst Arm repeat fails to resulting changes yielded silent mutations. These sites are stabilize endogenous b-catenin, yet can still induce axis already present in the analogous regions of the b-catenin duplication when expressed in Xenopus (Klymkowsky et cDNA. The creation of the A¯3 site in the plakoglobin al., 1999; Rubenstein et al., 1997). In addition, if cDNA allowed us to exchange the amino terminal portions of the molecules, while the creation of the NdeI site in the plakoglobin were acting to signal only in an indirect plakoglobin cDNA facilitated the exchange of sequences manner, it is dicult to understand how altering its following the Armadillo repeats. It should be noted that carboxyl terminus would enhance transactivation (Fig- neither swap changed amino acids in the Armadillo repeats, ure 4b). However, a possible explanation to this could be except for an MET to ILE change in the latter part of the that the carboxyl terminal region of plakoglobin serves armadillo repeat in swaps which exchanged the carboxyl to inhibit plakoglobin/LEF1/DNA complex formation termini of b-catenin and plakoglobin. (Zhurinsky et al., 2000). An answer to this question may Versions of b-catenin and plakoglobin tagged at the come from future studies in which plakoglobin con- carboxyl terminus with both V5 and His6 were created by structs are introduced into b-catenin-de®cient cells amplifying the coding sequences of each molecule to create (Haegel et al., 1995; Huelsken et al., 2000). fragments with 5' and 3' restriction sites but without stop codons. These fragments were cloned in-frame into the In conclusion, we have found several dierences that epitope tagging vector pCDNA6 (Invitrogen). Carboxyl may explain why mutations in b-catenin, but not terminal deletions of b-catenin were made using site-directed plakoglobin, are commonly found in human tumors. mutagenesis, creating stop codons at various sites in the One is that the observed point mutations in b-catenin, construct. Further details of all constructions are available but not analogous mutations in plakoglobin, confer upon request. added protein stability relative to the wild type version of the protein. Secondly, such `activated' versions of b- catenin are 4 ± 5 times more potent than plakoglobin in Cell culture and transient transfections the signaling assays we have described. In addition, b- catenin seems to bind with higher anity to two For each experiment, 6 cm plates were seeded with equal amounts of 293T cells on the night prior to transfection. Cells members of the LEF/TCF family of proteins, which were transfected by CaPO4 precipitation (Stratagene). For mediate the transcriptional eects of b-catenin and OT reporter assays, 1 mg of OT and 1 mg of pcDNA 3.1-lacZ possibly plakoglobin. were cotransfected with 2 mg of expression plasmid encoding various forms of b-catenin, plakoglobin, or b-catenin- plakoglobin chimera. Empty vector DNA was added to normalize the total amount of DNA in every transfection and Materials and methods each transfection was performed in duplicate. Thirty-six hours post-transfection, cells were harvested, extracts were Original plasmid sources prepared, and luciferase activity was determined (Promega Vectors containing cDNAs encoding the proteins used in this Luciferase Assay System). Readings were normalized for study were obtained from the following sources: b-catenin (P transfection eciency by measuring b-galactosidase activity Robbins), plakoglobin (W Franke), LEF1 (R Grosschedl), (as in Coso et al., 1995).

Oncogene Comparison of b-catenin and plakoglobin BO Williams et al 5727 Immunoprecipitation and Western blots 36 h after transfection were grown in DMEM without cysteine or methionine (supplemented with 10% dialysed Transfected 293T cells were harvested on ice with 1 ml of serum and glutamine) for 30 min. Cells were incubated in lysis buer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl 0.5% media containing 0.3 mCi ml of 35S translabel (ICN) for 2 h, NP-40, and protease inhibitors). Lysates were microcentri- labeling media was aspirated, and cells were grown in normal fuged at 14 000 r.p.m. for 10 min at 48C. For immunopre- growth media supplemented with 10 times the normal cipitations, lysates were normalized for total protein and concentration of cysteine and methionine (Sigma). At the incubated for 2 h at 48C, with 2 mg of anti-b-catenin indicated chase times, plates were lysed on ice using 1 ml of (Transduction Laboratories), anti-plakoglobin (Transduction 0.5% NP-40 lysis buer. Lysates were frozen at 7808C, Laboratories), or anti-V5 (Invitrogen) monoclonal antibodies. thawed, and spun at top speed in a microfuge for 10 min. Forty ml of anti-mouse IgG agarose beads (Sigma) were Each supernatant was removed to a new tube, 2 mg of anti- added, and samples were incubated for an additional hour at V5 antibody was added, and each sample was rocked at 48C 48C. Agarose beads were washed three times with 1 ml of for 2 h. Forty ml of a 50% slurry of anti-mouse IgG agarose 0.26 NP-40 lysis buer and bound proteins were eluted in (Sigma) was added to each tube, and samples were rocked for SDS sample buer. an additional hour at 48C. Pellets were washed three times Western blots were incubated at room temperature for 1 h with 1 ml of chilled NP-40 lysis buer. Thirty ml of SDS in blocking solution (0.1% Tween-20 in Tris buered saline loading buer was added to each pellet, tubes were boiled, and 5% dry milk). Blots were immunostained for B-catenin and samples were run out on a 10% Tris Glycine denaturing and plakoglobin with the following antibodies (Transduction polyacrylamide mini-gel (Novex). Gels were Coomasie Labs Anti-b-catenin (Catalog #C19220) and Anti-plakoglo- stained and destained using a solution of 20% methanol bin (Catalog #C26220); Anti-plakoglobin (Zymed Clone and 5% acetic acid to con®rm equal quantities of precipitated PG-11E4)). For Anti-V5 Westerns, blots were incubated for antibody in each sample. Gels were incubated in Enlightening 1 h in anti-V5-HRP antibody (Invitrogen) diluted 1 : 2000 in enhancement solution for 30 min, dried onto Whatman blocking solution, washed three times in TBS-Tween, paper, and exposed to ®lm. subjected to ECL, and exposed to ®lm. For Anti-myc Westerns, blots were incubated for 1 h in mouse anti-myc monoclonal antibody (Santa Cruz) diluted 1 : 1000 in blocking solution, washed three times in TBS-Tween, incubated in anti-mouse IgG-peroxidase (Boehringer Man- nheim) diluted 1 : 10 000 in blocking solution for 1 h at room Acknowledgments temperature, washed three times in TBS-Tween, exposed to We would like to thank Mario Chamorro for advice on the ECL (Amersham), and exposed to ®lm. pulse/chase analysis and other members of the Varmus laboratory for helpful discussions. BO Williams was a post- doctoral fellow of the Damon Runyon-Walter Winchell Pulse/chase experiments Cancer Research Fund. GD Barish was a Howard Hughes 293T cells were transfected (as previously detailed) with V5 Medical Institute-NIH Research Scholar. MW Klymkowsky epitope tagged b-catenin or plakoglobin plasmid DNA, and was supported by NIH grant GM54001.

References

Aberle H, Bierkamp C, Torchard D, Serova O, Wagner T, Crawford HC, Fingleton BM, Rudolph-Owen LA, Goss KJ, Natt E, Wirsching J, Heidkamper C, Montagna M, Lynch Rubinfeld B, Polakis P and Matrisian LM. (1999). HT, Lenoir GM, Scherer G, Feunteun J and Kemler R. Oncogene, 18, 2883 ± 2891. (1995). Proc. Natl. Acad. Sci. USA, 92, 6384 ± 6388. Dale TC. (1998). Biochem. J., 329, 209 ± 232. Aberle H, Schwartz H and Kemler R. (1996). J. Cell. Denk C, Hulsken J and Schwarz E. (1997). Cancer Lett., 120, Biochem., 61, 514 ± 523. 185 ± 193. Barker N, Morin PJ and Clevers H. (2000). Adv. Cancer Res., Fagotto F, Gluck U and Gumbiner BM. (1998). Curr. Biol., 77, 1±24. 8, 181 ± 190. Barth AI, Nathke IS and Nelson WJ. (1997). Curr. Opin. Cell Fagotto F, Jho E, Zeng L, Kurth T, Joos T, Kaufmann C and Biol., 9, 683 ± 690. Costantini F. (1999). J. Cell. Biol., 145, 741 ± 756. Behrens J, Jerchow BA, Wurtele M, Grimm J, Asbrand C, Fan MJ, Gruning W, Walz G and Sokol SY. (1998). Proc. Wirtz R, Kuhl M, Wedlich D and Birchmeier W. (1998). Natl. Acad. Sci. USA., 95, 5626 ± 5631. Science, 280, 596 ± 599. Farr III GH, Ferkey DM, Yost C, Pierce SB, Weaver C and Behrens J, von Kries JP, Kuhl M, Bruhn L, Wedlich D, Kimelman D. (2000). J. Cell. Biol., 148, 691 ± 702. Grosschedl R and Birchmeier W. (1996). Nature, 382, Gelderloos J, Witcher L, Cowin P and Klymkowsky MW. 638 ± 642. (1997). Cytoskeletal-membrane interactions and signal Bierkamp C, Schwarz H, Huber O and Kemler R. (1999). transduction. Landes Bioscience, Austin, Texas, pp. 13 ± Development, 126, 371 ± 381. 30. Brannon M, Gomperts M, Sumoy L, Moon RT and Gradl D, Kuhl M and Wedlich D. (1999). Mol. Cell. Biol., 19, Kimelman D. (1997). Genes Dev., 11, 2359 ± 2370. 5576 ± 5587. Caca K, Kolliga FT, Ji X, Hayes M, Qian J, Yahanda A, Haegel H, Larue L, Ohsugi M, Fedorov L, Herrenknecht K Rimm DL, Costa J and Fearon E. (1999). Cell. Growth and Kemler R. (1995). Development, 121, 3529 ± 3537. Dier., 10, 369 ± 376. HartM,ConcordetJP,LassotI,AlbertI,dellosSantosR, Carnac G, Kodjabachian L, Gurdon JB and Lemaire P. Durand H, Perret C, Rubinfeld B, Margottin F, Benarous (1996). Development, 122, 3055 ± 3065. R and Polakis P. (1999). Curr. Biol., 9, 207 ± 210. Chan EF, Gat U, McNi JM and Fuchs E. (1999). Nat. Hart MJ, de los Santos R, Albert IN, Rubinfeld B and Genet., 21, 410 ± 413. Polakis P. (1998). Curr. Biol., 8, 573 ± 581. Cook D, Fry MJ, Hughes K, Sumathipala R, Woodgett JR He TC, Sparks AB, Rago C, Hermeking H, Zawel L, da and Dale TC. (1996). EMBO J., 15, 4526 ± 4536. Costa L, Morin PJ, Vogelstein B and Kinzler KW. (1998). Coso OA, Chiariello M, Yu JC, Teramoto H, Crespo P, Xu Science, 281, 1509 ± 1512. N, Miki T and Gutkind JS. (1995). Cell., 81, 1137 ± 1146.

Oncogene Comparison of b-catenin and plakoglobin BO Williams et al 5728 Hecht A, Litterst CM, Huber O and Kemler R. (1999). J. Nusse R and Varmus HE. (1992). Cell., 69, 1073 ± 1087. Biol. Chem., 274, 18017 ± 18025. Ogawa K, Yamada Y, Kishibe K, Ishizaki K and Tokusashi Hsu SC, Galceran L and Grosschedl R. (1998). Mol. Cell. Y. (1999). Cancer Res., 59, 1830 ± 1833. Biol., 18, 4807 ± 4818. Orsulic S and Peifer M. (1996). J. Cell. Biol., 134, 1283 ± Huang H, Fujii H, Sankila A, Mahler-Araujo BM, Matsuda 1300. M, Cathomas G and Ohgaki H. (1999). Am.J.Pathol., Pantel K, Passlick B, Vogt J, Stosiek P, Angstwurn M, Seen- 155, 1795 ± 1801. Hibler R, Haussinger K, Thetter O, Izbicki JR and Huber AH, Nelson WJ and Weis WI. (1997). Cell., 90, 871 ± Riethmuller G. (1998). J. Clin. Oncol., 16, 1407 ± 1413. 882. Papko J, Rubinfeld B, Schryver B and Polakis P. (1996). Huber O, Korn R, McLaughlin J, Ohsugi M, Herrmann BG Mol. Cell. Biol., 16, 2128 ± 2134. and Kemler R. (1996). Mech. Dev., 59, 3±10. Polakis P. (1999). Curr.Opin.Genet.Dev.,9, 15 ± 21. Huelsken J, Vogel R, Brinkmann V, Erdmann B, Birchmeier Por®ri E, Rubinfeld B, Albert I, Hovanes K, Waterman M C and Birchmeier W. (2000). J. Cell. Biol., 148, 567 ± 578. and Polakis P. (1997). Oncogene, 15, 2833 ± 2839. Ikeda S, Kishida S, Yamamoto H, Murai H, Koyama S and Rubenstein A, Merriam J and Klymkowsky MW. (1997). Kikuchi A. (1998). EMBO J., 17, 1371 ± 1384. Dev. Genet., 20, 91 ± 102. Jiang J and Struhl G. (1998). Nature, 391, 493 ± 496. Rubinfeld B, Robbins P, El-Gamil M, Albert I, Por®ri E and Kessler DS. (1997). Proc. Natl. Acad. Sci. USA, 94, 13017 ± Polakis P. (1997). Science, 275, 1790 ± 1792. 13022. Rubinfeld B, Souza B, Albert I, Muller O, Chamberlain SH, Kikuchi A. (1999a). Cytokine Growth Factor Rev., 10, 255 ± Masiarz FR, Munemitsu S and Polakis P. (1993). Science, 265. 262, 1731 ± 1734. Kikuchi A. (1999b). Cell. Signal, 11, 777 ± 788. Sadot E, Simcha I, Iwai K, Ciechanover A, Geiger B and Kitagawa M, Hatakeyama S, Shirane M, Matsumoto M, Ben-Ze'ev A. (2000). Oncogene, 19, 1992 ± 2001. Ishida N, Hattori K, Nakamichi I, Kikuchi A and Sakanaka C, Weiss JB and Williams LT. (1998). Proc. Natl. Nakayama K. (1999). EMBO J., 18, 2401 ± 2410. Acad.Sci.USA,95, 3020 ± 3023. Klymkowsky MW, Williams BO, Barish GD, Varmus HE Simcha I, Geiger B, Yehuda-Levenberg S, Salomon D and and Vourgourakis YE. (1999). Mol. Biol. Cell., 10, 3151 ± Ben-Ze'ev A. (1996). J. Cell. Biol., 133, 199 ± 209. 3169. Simcha I, Shtutman M, Salomom D, Zhurinsky J, Sadot E, Kodama S, Ikeda S, Asahara T, Kishida M and Kikuchi A. Geiger B and Ben-Ze'ev A. (1998). J. Cell Biol., 141, (1999). J. Biol. Chem., 274, 27682 ± 27688. 1433 ± 1448. Kolligs FT, Kolligs B, Hajra KM, Hu G, Tani M, Cho KR Smalley MJ, Sara E, Paterson H, Naylor S, Cook D, and Fearon ER. (2000). Genes Dev., 14, 1319 ± 1331. Jayatilake H, Fryer LG, Hutchinson L, Fry MJ and Dale Kondo Y, Kanai Y, Sakamoto M, Genda T, Mizokami M, TC. (1999). EMBO J, 18, 2823 ± 2835. Ueda R and Hirohashi S. (1999). Jpn. J. Cancer Res., 90, Sparks AB, Morin PJ, Vogelstein B and Kinzler KW. (1998). 1301 ± 1309. Cancer Res., 58, 1130 ± 1134. Korinek V, Barker N, Morin PJ, van Wichen D, de Weger R, Su LK, Vogelstein B and Kinzler KW. (1993). Science, 262, Kinzler KW, Vogelstein B and Clevers H. (1997). Science, 1734 ± 1737. 275, 1784 ± 1787. Tao YS, Edwards RA, Tubb B, Wang S, Bryant J and Kowalczyk AP, Bornslaeger EA, Norvell SM, Palka HL and McCrea PD. (1996). J. Cell. Biol., 134, 1271 ± 1281. Green KJ. (1999). Int. Rev. Cytol., 185, 237 ± 302. Tetsu O and McCormick F. (1999). Nature, 398, 422 ± 426. Latres E, Chiaur DS and Pagano M. (1999). Oncogene, 18, van de Wetering M, Cavallo R, Dooijes D, van Beest M, van 849 ± 854. Es J, Loureiro J, Ypma A, Hursh D, Jones T, Bejsovec A, Laurent MN, Blitz IL, Hashimoto C, Rothbacher U and Cho Peifer M, Mortin M and Clevers H. (1997). Cell, 88, 789 ± KW. (1997). Development, 124, 4905 ± 4916. 799. Legoix P, Bluteau O, Bayer J, Perret C, Balabaud C, Belghiti Wahl J, Sacco P, McGranahan ST, Sauppe L, Wheelock M J, Franco D, Thomas G, Laurent-Puig P and Zucman- and Johnson K. (1996). J. Cell. Sci., 109, 1143 ± 1154. Rossi J. (1999). Oncogene, 18, 4044 ± 4046. White P, Aberle H and Vincent JP. (1998). J. Cell. Biol., 140, Liu C, Kato Y, Zhang Z, Do VM, Yankner BA and He X. 183 ± 195. (1999). Proc. Natl. Acad. Sci. U S A, 96, 6273 ± 6278. Winston JT, Strack P, Beer RP, Chu CY, Elledge SJ and Margottin F, Bour SP, Durand H, Selig L, Benichou S, Harper JW. (1999). Genes Dev., 13, 270 ± 283. Richard V, Thomas D, Strebel K and Benarous R. (1998). Witcher LL, Collins R, Puttagunta S, Mechanic SE, Munson Mol. Cell., 1, 565 ± 574. M, Gumbiner B and Cowin P. (1996). J. Biol. Chem., 271, Marikawa Y and Elinson RP. (1998). Mech. Dev., 77, 75 ± 80. 10904 ± 10909. McKendry R, Hsu SC, Harland RM and Grosschedl R. Yamamoto H, Kishida S, Uochi T, Ikeda S, Koyama S, (1997). Dev. Biol., 192, 420 ± 431. Asashima M and Kikuchi A. (1998). Mol. Cell. Biol., 18, Miller JR and Moon RT. (1997). J. Cell. Biol., 139, 229 ± 243. 2867 ± 2875. Miyoshi Y, Iwao K, Nagasawa Y, Aihara T, Sasaki Y, Yokoya F, Imamoto N, Tachibana T and Yoneda Y. (1999). Imaoka S, Murata M, Shimano T and Nakamura Y. Mol. Biol. Cell., 10, 1119 ± 1131. (1998). Cancer Res., 58, 2524 ± 2527. Yost C, Torres M, Miller JR, Huang E, Kimelman D and Molenaar M, van de Wetering M, Oosterwegel M, Peterson- Moon RT. (1996). Genes Dev, 10, 1443 ± 1454. Maduro J, Godsave S, Korinek V, Roose J, Destree O and Zeng L, Fagotto F, Zhang T, Hsu W, Vasicek TJ, Perry III Clevers H. (1996). Cell., 86, 391 ± 399. WL, Lee JJ, Tilghman SM, Gumbiner BM and Costantini Morin PJ, Sparks AB, Korinek V, Barker N, Clevers H, F. (1997). Cell, 90, 181 ± 192. Vogelstein B and Kinzler KW. (1997). Science, 275, 1787 ± Zhurinsky J, Shtutman M and Ben Ze'ev A. (2000). Mol. Cell 1790. Biol., in press. Munemitsu S, Albert I, Rubinfeld B and Polakis P. (1996). Zorn AM, Barish GD, Williams BO, Lavender P, Klym- Mol. Cell. Biol., 16, 4088 ± 4094. kowsky MW and Varmus HE. (1999). Mol. Cell., 4, 487 ± NhieuJT,RenardCA,WeiY,CherquiD,ZafraniESand 498. Buendia MA. (1999). Am. J. Pathol., 155, 703 ± 710.

Oncogene