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doi: 10.1242/10.1242/jcs.00154

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Protein kinase CKII regulates the interaction of ß-catenin with -catenin and its protein stability

Stephan Bek^* and Rolf Kemler

Department of Molecular Embryology, Max-Planck Institute of Immunobiology, Stuebeweg 51, D-79108 Freiburg, Germany
^* Present address: Stephan Bek, Aventis Pharma Deutschland, Functional Genomics, Industriepark Hoechst, G879/029, D-65926 Frankfurt/Main, Germany

View larger version (25K): [in a new window]   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).

View larger version (45K): [in a new window]   Fig. 2. CKII phosphorylates the N-terminus of ß-catenin. (A) ß-catenin wt and deletion constructs expressed as GST-fusion proteins and tested in kinase assays in vitro. (B) The proteins were subjected to kinase assays as described in Fig. 1A, but with or without heparin (5 µg/µl) as an inhibitor of CKII-activity. Phosphorylation is efficiently reduced by heparin at the N-terminus (aa 1-119, 1-302) but not at the C-terminus of ß-catenin. In the control-lane (co), unrelated mouse IgG in the initial immunoprecipitation was used and incubated with full-length GST ß-catenin. The Coomassie-stain provides the loading control for the respective fusion-proteins. (C) Three conserved CKII consensus motifs can be found in the N-terminus of ß-catenin (aa 1-119). These sites are located in close proximity to the GSK-3ß consensus motif (Aberle et al., 1997). No additional CKII sites are located in the extended N-terminal region (aa 120-302) of ß-catenin (not shown).

View larger version (34K): [in a new window]   Fig. 3. Mutations in CKII consensus-motifs stabilize ß-catenin. (A) 293 cells stably transfected with either wt or Ser/Thr-mutant HA-ß-catenin were subjected to pulse-chase experiments as described in Materials and Methods. The triple-mutant used contained the three mutations indicated in panel A. Equal amounts of incorporated radioactivity were immunoprecipitated with anti-HA antibodies at the time points indicated and the autoradiographs were quantified by phospho-imager analysis. The half-life of Ser/Thr-mutant ß-catenin was approximately two- to threefold that of wt ß-catenin. (B) Quantitative measurements of band intensities from three independent experiments, using MacBas image analyzing software (circles, wild-type ß-catenin; squares, mutant ß-catenin).

View larger version (36K): [in a new window]   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.

View larger version (50K): [in a new window]   Fig. 4. CKII associates with components of the cytoplasmic degradation complex and modulates the ß-catenin-Axin interaction. (A) 293 cells were transiently transfected with Myc-tagged Axin or with Myc-GSK-3ß (empty vectors for control, lanes 1 and 3) and endogenous CKII was immunoprecipitated from comparable amounts of whole-cell lysates. Both Myc-Axin (lane 2) and Myc-GSK-3ß (lane 4) were detected in the immunoprecipitates. (B) HA-tagged wt or Ser/Thr-mutated (single or triple mutation) ß-catenin were transfected together with Myc-tagged Axin into 293 cells. The expression of Myc-Axin was controlled by immunoblot (lanes 4-6). Comparable amounts of Myc-Axin were immunoprecipitated with anti-Myc antibodies and the immunoprecipitates were probed with anti-HA. The association of HA-ß-catenin to Myc-Axin is reduced to an extent that depends on the number of introduced mutations in the CKII consensus motifs (lanes 1-3).

View larger version (28K): [in a new window]   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.

View larger version (56K): [in a new window]   Fig. 7. Ser/Thr-mutant ß-catenin enhances cell migration. (A) Stable transfectants of 293 cells expressing HA-tagged wt or Ser/Thr-mutant ß-catenin containing all three mutations, were subjected to wound healing experiments. Wound closure was much enhanced in cells expressing Ser/Thr-mutant ß-catenin suggesting an increased migratory potential of the cells. (B) Cells were treated as described in A, but stained with antibodies for the proliferation marker Ki-6. The morphology of the cells is documented with brightfield-photography. No significant difference in cell proliferation was observed between the two cell types. (C) Stable transfectants of 293 cells expressing HA-tagged wt or Ser/Thr-mutant ß-catenin were stained for HA ß-catenin and -catenin with indirect immunofluorescence. Cells expressing HA wt ß-catenin showed a stronger staining at cell-cell-contact sites compared with cells expressing HA Ser/Thr-mutant ß-catenin. The same is true for the distribution of -catenin in both cell-lines. However, significant amounts of -catenin in mutant cells are still localized at the membrane due to the association with endogenous ß-catenin.

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