A comparative evaluation of b-catenin and plakoglobin signaling activity Bart O Williams* ,1,3,5 , Grant D Barish 1,5 , Michael W Klymkowsky 2 and Harold E Varmus 1,4 1 National Cancer Institute, Division of Basic Sciences, National Institutes of Health, Bethesda, Maryland, MD 20892, USA; 2 Molecular, Cellular & Developmental Biology, University of Colorado Boulder, Boulder, Colorado, CO 80309-0347, USA Vertebrates have two Armadillo-like proteins, b-catenin and plakoglobin. Mutant forms of b-catenin with oncogenic activity are found in many human tumors, but plakoglobin mutations are not commonly found. In fact, plakoglobin has been proposed to suppress tumorigenesis. To assess dierences between b-catenin and plakoglobin, we compared several of their biochem- ical properties. After transient transfection of 293T cells with an expression vector encoding either of the two proteins, soluble wild type b-catenin does not signi®- cantly accumulate, whereas soluble wild type plakoglobin is readily detected. As anticipated, b-catenin is stabilized by the oncogenic mutation S37A; however, the analogous mutation in plakoglobin (S28A) does not alter its half- life. S37A-b-catenin activates a TCF/LEF-dependent reporter 20-fold more potently than wild type b-catenin, and *5-fold more potently than wild type or S28A plakoglobin. These dierences may be attributable to an enhanced anity of S37A b-catenin for LEF1 and TCF4, as observed here by immunoprecipitation assays. We show that the carboxyl-terminal domain is largely responsible for the dierence in signaling and that the Armadillo repeats account for the remainder of the dierence. The relatively weak signaling by plakoglobin and the failure of the S28A mutation to enhance its stability, may explain why plakoglobin mutations are infrequent in malignancies. Oncogene (2000) 19, 5720 ± 5728. Keywords: b-catenin; plakoglobin; Wnt; LEF/TCF Introduction Wnts were originally identi®ed as key regulators of early development in Drosophila and as proto- oncogenes in mammals (Nusse and Varmus, 1992). Wnts are secreted polypeptides that bind to members of the Frizzled receptor family, triggering a series of cytosolic events that increase the stability of the armadillo protein (Drosophila) or its homologs in higher vertebrates (b-catenin and plakoglobin, or Armadillo-like proteins [ALPs]), ultimately leading to changes in gene expression (reviewed in Dale, 1998). Subsequent studies have begun to resolve the molecular mechanism underlying regulation of these ALP's by Wnt signaling. Much of what is known about their regulation is based on studies with b- catenin. When not bound to cadherins (Aberle et al., 1996), b-catenin is targeted for degradation via phosphorylation by the constitutively active glycogen synthase kinase 3 (GSK3) (reviewed in Dale, 1998). GSK3 forms a complex with the product of the tumor suppressor gene adenomatous polyposis coli (APC) and with axin, thereby targeting b-catenin for ubiquitin-dependent proteolysis (Behrens et al., 1998; Farr et al., 2000; Hart et al., 1998; Ikeda et al., 1998; Sakanaka et al., 1998; Yamamoto et al., 1998; Zeng et al., 1997), mediated by the F-box protein Slimb/ b- TrCP (Hart et al., 1999; Jiang and Struhl, 1998; Kitagawa et al., 1999; Latres et al., 1999; Liu et al., 1999; Margottin et al., 1998; Marikawa and Elinson, 1998; Winston et al., 1999). In the presence of a Wnt signal, GSK3 activity is inhibited, and cytosolic levels of b-catenin rise (Cook et al., 1996; Dale, 1998). Cytosolic b-catenin enters the nucleus where it can heterodimerize with members of the T-Cell Factor/ Lymphoid Enhancer Factor-1 (TCF/LEF) subfamily of high mobility group (HMG) box DNA binding proteins (Behrens et al., 1996; Huber et al., 1996; Molenaar et al., 1996; van de Wetering et al., 1997). Binding of b-catenin alters the activity of TCFs, thereby aecting target gene transcription (reviewed in Barker et al., 2000). A number of transcriptional targets for Wnt signaling have been identi®ed, including the dorsalizing genes Siamois (Brannon et 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 (He et al., 1998) and cyclin D1 (Tetsu and McCormick, 1999); ®bronectin (Gradl et al., 1999); and the gene encoding the matrix metalloprotease matrilysin (Crawford et al., 1999). In addition to their interactions with TCF/LEFs, ALPs also serve to anchor actin ®laments to adherens junctions by binding simultaneously to classical cadherins and to the actin-binding protein a-catenin (reviewed in Aberle et al., 1996). Vertebrates contain a second type of cadherin-based adherens junction, the desmosome, which serves to anchor the intermediate ®lament network to the plasma membrane (Kowalczyk et al., 1999). Plakoglobin is specialized to bind to desmosomal cadherins (Gelderloos et al., 1997; Wahl et 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 plakoglobin (Bierkamp et al., 1999). Binding of plakoglobin to desmosomal cadherins blocks plakoglo- bin's ability to bind to a-catenin, thereby inhibiting the interaction of actin ®laments (Gelderloos et al., 1997). Drosophila does not appear to have cytoplasmic intermediate ®laments, and therefore has only a single Oncogene (2000) 19, 5720 ± 5728 ã 2000 Macmillan Publishers Ltd All rights reserved 0950 ± 9232/00 $15.00 www.nature.com/onc *Correspondence: BO Williams Current addresses: 3 Van Andel Research Institute 333 Bostwick NE Grand Rapids, MI 49503, USA; 4 Memorial Sloan-Kettering Cancer Center, 75 York Avenue, New York, NY 10021, USA 5 These two authors contributed equally to this work Received 9 June 2000; revised 8 September 2000; accepted 13 September 2000