Fig. 5. Synergistic binding of CKII and GSK-3ß to ß-catenin. GST-fused wt or Ser/Thr-mutant ß-catenin coupled to GSH-Sepharose (500 ng each lane) was phosphorylated with either recombinant CKII or GSK-3ß, or both, according to the instructions of the manufacturers, and bound kinases were detected in immunoblots with anti-CKII (A) or anti-GSK-3ß-antibodies (B). Equal amounts of recombinant ß-catenin were used as shown with anti-GST antibodies (C). In parallel experiments (D) the reaction mixture contained ³²P-ATP and gels were autoradiographed for 8 hours. CKII binds efficiently to wt GST-ß-catenin (A, lanes 3,5), whereas binding to the Ser/Thr-mutant protein is reduced (A, lanes 7,9). GSK-3ß bound equally well to wt and Ser/Thr-mutant ß-catenin (B, lanes 4,8). However, binding of GSK-3ß to wt ß-catenin is clearly enhanced when ß-catenin was pre-incubated with CKII (B, lanes 4,5). The lower molecular weight bands appearing in lanes 3, 5 and 7 are probably due to crossreactivity of the GSK-3ß antibody with CKII-. Phosphorylation of wt ß-catenin is significantly enhanced when both kinases were consecutively used (D, lane 5). Ser/Thr-mutant ß-catenin is not phosphorylated by CKII (D, lane 7) and no difference in phosphate-incorporation is observed in lanes 8 and 9.