Fig. 6. CKII regulates binding of ß-catenin to -catenin. (A) 293 cells stably expressing wt or Ser/Thr-mutant HA-tagged ß-catenin were grown to 80% confluency and, for subcellular fractionation, cells were subjected to sequential detergent extractions (see Materials and Methods). The Ser/Thr-mutant form of ß-catenin contained all three mutations indicated in Fig. 3A. From each fraction HA-ß-catenin immunoprecipitates were immunoblotted with anti-HA-antibodies. The same procedure was performed with untransformed 293 cells, which express only endogenous ß-catenin. Wt ß-catenin from these preparations was immunoprecipitated and immunoblotted with anti ß-catenin antibodies. Compared with wt ß-catenin and endogenous ß-catenin, more Ser/Thr-mutant ß-catenin was found in the cytosolic fraction, whereas the amount in the insoluble cytoskeletal fraction was significantly reduced. (B) 500 ng of either GST wt or Ser/Thr-mutant ß-catenin coupled to GSH-Sepharose was incubated with recombinant CKII in the absence of ATP. After several washes His₆-tagged -catenin was added under association-conditions (see Materials and Methods) and bound proteins were probed for -catenin or GST in immunoblots. The affinity of -catenin to Ser/Thr-mutant ß-catenin clearly decreased compared with wt ß-catenin. (C) GST-proteins were pre-phosphorylated with CKII in the presence of 20 mM ATP prior to the incubation with -catenin. Binding of His₆-tagged -catenin to pre-phosphorylated wt ß-catenin was significantly increased but no phosphorylation-dependent difference for binding of -catenin was observed with Ser/Thr-mutant ß-catenin.

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