The adenomatous polyposis coli gene (APC) is mutated in most colon cancers. The APC protein binds to the cellular adhesion molecule beta-catenin, which is a mammalian homolog of ARMADILLO, a component of the WINGLESS signaling pathway in Drosophila development. Here it is shown that when beta-catenin is present in excess, APC binds to another component of the WINGLESS pathway, glycogen synthase kinase 3beta (GSK3beta), a mammalian homolog of Drosophila ZESTE WHITE 3. APC was a good substrate for GSK3 beta in vitro, and the phosphorylation sites were mapped to the central region of APC. Binding of beta-catenin to this region was dependent on phosphorylation by GSK3 beta.

Signal transduction by beta-catenin involves its posttranslational stabilization and downstream coupling to the Lef and Tcf transcription factors. Abnormally high amounts of beta-catenin were detected in 7 of 26 human melanoma cell lines. Unusual messenger RNA splicing and missense mutations in the beta-catenin gene (CTNNB1) that result in stabilization of the protein were identified in six of the lines, and the adenomatous polyposis coli tumor suppressor protein (APC) was altered or missing in two others. In the APC-deficient cells, ectopic expression of wild-type APC eliminated the excess beta-catenin. Cells with stabilized beta-catenin contained a constitutive beta-catenin-Lef-1 complex. Thus, genetic defects that result in up-regulation of beta-catenin may play a role in melanoma progression.

The APC suggests that deregulation of cell adhesion may be involved. Calcium-dependent cell-cell adhesion is maintained by interactions between transmembrane cadherin molecules which require the association of catenins with their cytoplasmic domains (8-10). The stability of the (3-catenincadherin complex is in turn modulated by a posttranscriptional mechanism that affects the relative stability of ,B-catenin itself (11). This mechanism is engaged by the product of the WNT1 oncogene which promotes the accumulation of (3-and ly-catenins and strengthens calcium-dependent cell adhesion (11,12). In addition to simply supporting cell adhesion, a role for P-catenin in signal transduction has also been proposed. In Drosophila, the appearance of armadillo, the f3-catenin homolog, in the cytoplasm is dependent upon expression of wingless, the WNT1 homolog (13 Lipofectin (BRL) was added to the washed cells. Transfection medium was removed after 20-24 hr and 2 ml of growth medium (Leibovitz L-15 medium with penicillin, streptomycin, L-glutamine, and 10% fetal bovine serum; Irvine Scientific) was added to each well. Twenty-four hours later cells were harvested or were analyzed by immunofluorescence. Detection of 83-galactosidase expression was performed by the protocol published by Stratagene.Immunological Procedures. The general procedures for immunofluorescence analysis, microscopy, and photography have been described (15). Transfected cells were analyzed 48 hr after transfection. Cells were grown on coverslips, washed with phosphate-buffered saline, and then fixed in methanol at -20°C. After blocking in 10% powdered milk solution, detection of 3-catenin was performed with either a 1:200 dilution of rabbit polyclonal anti-,B-catenin serum (gift of B. Gumbiner, Sloan-Kettering) or, in the case of costaining experiments (see Fig. 2C), a 1:50 dilution of a mouse monoclonal antibody (Transduction Laboratories, Lexington, KY). APC protein was detected with affinity-purified rabbit polyclonal antibodies (6). For secondary antibodies, fluorescein-conjugated goatanti-rabbit serum (Sigma) or Texas Red-conjugated donkey anti-mouse antibodies (Cappel) were used at dilutions of 1:32 and 1:60, respectively. Except where noted, all SDS/PAGE was performed with 8% polyacrylamide gels. Protein blots were developed overnight with the following antibodies: a 1:2500 dilution of rabbit polyclonal anti-/3-catenin serum (B. Gumbiner); a 1:5000 dilution of anti-p120GAP (GTPaseactivating protein) serum (16), or a 1:500 dilution of anti-acatenin serum (J. Papkov, Sugen, Redwood City, CA), or a mixture of affinity-purified anti-APC2 and anti-APC3 antibodies (6), each at 0.2 ,ug/ml. After a 1-hr incubation in 25 mM Tris-buffered saline containing 0.05% Tween 20 and 125I1 labeled protein A (Amersham) at 1 ,uCi/ml (1 ,uCi = 37 kBq) blots were washed, exposed to x-ray film, and then quantitated by a 12-hr exposure on an Ambis model 4000 3 scanner. The ECL system (Amersham) was used for detection of the protein blots shown in Figs. 4 B and 5 A an...

Mutations in the human APC gene are linked to familial adenomatous polyposis and to the progression of sporadic colorectal and gastric tumors. To gain insight into APC function, APC-associated proteins were identified by immunoprecipitation experiments. Antibodies to APC precipitated a 95-kilodalton protein that was purified and identified by sequencing as beta-catenin, a protein that binds to the cell adhesion molecule E-cadherin. An antibody specific to beta-catenin also recognized the 95-kilodalton protein in the immunoprecipitates. These results suggest that APC is involved in cell adhesion.

Axin acts as a scaffold upon which APC, beta-catenin and GSK3 beta assemble to coordinate the regulation of beta-catenin signaling.

Defects in beta-catenin regulation contribute to the neoplastic transformation of mammalian cells. Dysregulation of beta-catenin can result from missense mutations that affect critical sites of phosphorylation by glycogen synthase kinase 3beta (GSK3beta). Given that phosphorylation can regulate targeted degradation of beta-catenin by the proteasome, beta-catenin might interact with an E3 ubiquitin ligase complex containing an F-box protein, as is the case for certain cell cycle regulators. Accordingly, disruption of the Drosophila F-box protein Slimb upregulates the beta-catenin homolog Armadillo. We reasoned that the human homologs of Slimb - beta-TrCP and its isoform beta-TrCP2 (KIAA0696) - might interact with beta-catenin. We found that the binding of beta-TrCP to beta-catenin was direct and dependent upon the WD40 repeat sequences in beta-TrCP and on phosphorylation of the GSK3beta sites in beta-catenin. Endogenous beta-catenin and beta-TrCP could be coimmunoprecipitated from mammalian cells. Overexpression of wild-type beta-TrCP in mammalian cells promoted the downregulation of beta-catenin, whereas overexpression of a dominant-negative deletion mutant upregulated beta-catenin protein levels and activated signaling dependent on the transcription factor Tcf. In contrast, beta-TrCP2 did not associate with beta-catenin. We conclude that beta-TrCP is a component of an E3 ubiquitin ligase that is responsible for the targeted degradation of phosphorylated beta-catenin.

No abstract

Rap1 is a small Ras‐related GTPase which when over‐expressed is able to revert transformation by Ki‐Ras. We have investigated the role of Rap1 in regulating ‘normal’ Ras function by studying the activation of the mitogen‐activated protein (MAP) kinases ERK1 and ERK2 by two fundamentally different growth factors, epidermal growth factor (EGF) and 1‐oleoyl‐lyso‐phosphatidic acid (LPA). Conditional expression of RasN17 (a dominant‐negative mutant) in Rat‐1 cells inhibited activation of MAP kinases by EGF and also LPA, the first time a defined G‐protein‐coupled receptor mitogen has been shown to require Ras to exert its effects. Conditional or constitutive expression of even low levels of RapV12 (a mutant insensitive to Rap‐GAP) attenuated activation of MAP kinases by EGF and LPA, but did not interfere with growth factor‐stimulated increases in Ras‐GTP, indicating that signalling from receptors to Ras was not impaired. Inhibition of Ras‐mediated signalling with either RasN17 or RapV12 attenuated DNA synthesis by EGF and LPA. We conclude that receptor tyrosine kinases and G‐protein‐coupled receptors use Ras as a common step in signalling to MAP kinases and that Rap‐GTP (RapV12) at physiological levels interferes with downstream signalling from Ras to MAP kinases in vivo.