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.

Structural features of v‐kit, the oncogene of HZ4 feline sarcoma virus, suggested that this gene arose by transduction and truncation of cellular sequences. Complementary DNA cloning of the human proto‐oncogene coding for a receptor tyrosine kinase confirmed this possibility: c‐kit encodes a transmembrane glycoprotein that is structurally related to the receptor for macrophage growth factor (CSF‐1) and the receptor for platelet‐derived growth factor. The c‐kit gene is widely expressed as a single, 5‐kb transcript, and it is localized to human chromosome 4 and to mouse chromosome 5. A c‐kit peptide antibody permitted the identification of a 145,000 dalton c‐kit gene product that is inserted in the cellular plasma membrane and is capable of self‐phosphorylation on tyrosine residues in both human glioblastoma cells and transfected mouse fibroblasts. Our results suggest that p145c‐kit functions as a cell surface receptor for an as yet unidentified ligand. Furthermore, carboxy‐ and amino‐terminal truncations that occurred during the viral transduction process are likely to have generated the transformation potential of v‐kit.

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.

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