Fig. 1. CKII interacts with and phosphorylates ß-catenin. (A) ß-catenin immunoprecipitates were collected from cell lysates of epithelial CMT cells, and associated kinases were eluted and tested for activity against GST ß-catenin in kinase assays in vitro. Competitor peptides containing CKII-consensus motifs significantly decreased phosphorylation of GST ß-catenin compared with control-peptides (co), both at 0.5 mM (compare lanes 4 and 6). Other controls included unrelated mouse IgG in the initial immunoprecipitation (lane 1) and GST (lane 2). The Coomassie-stained gel of the autoradiography is shown. (B) Association of CKII and ß-catenin in vivo. CKII- was immunoprecipitated from cell lysates of 293 and NIH 3T3 cells and in each case ß-catenin was detected by immunoblotting of the immunoprecipitates (lanes 2,4). To demonstrate the specifity of the association, protein kinase A (PkA) was immunoprecipitated and blotted against ß-catenin in both cases (lanes 1,3). ß-catenin could not be detected in the PkA-precipitates. (C) In a reverse experiment, Myc-tagged CKII- or the Myc-tagged version of ERK-2 were transiently expressed in 293 cells and after 48 hours immunoprecipitates were collected with anti-ß-catenin and probed with anti-myc antibodies. Transfected or control lysates were tested in immunoblot for the correct expression of Myc-ERK2 (lane 1) or CKII (lane 2). Whereas Myc-ERK2 was not found in ß-catenin immunoprecipitates (lane 3), Myc-CKII co-precipitates with ß-catenin in these experiments (lane 4). (D) GST pulldown assays with recombinant ß-catenin and recombinant CKII- were performed in combination as indicated. CKII- was pre-adsorbed with GSH-beads before use; the overall amount of CKII- is shown in lane 1 (input). Proteins where allowed to associate in the presence or absence of ATP under phosphorylation-conditions described in Materials and Methods. GST ß-catenin was precipitated with GSH-beads and associated CKII- was detected by immunoblotting. Direct binding of CKII- to ß-catenin is enhanced in the presence of a phosphate-donor (compare lanes 5 and 6).