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Regulation of S33/S37 phosphorylated ß-catenin in normal and transformed cells

Einat Sadot^1,*, Maralice Conacci-Sorrell¹, Jacob Zhurinsky¹, Dalia Shnizer², Zeev Lando², Dorit Zharhary², Zvi Kam¹, Avri Ben-Ze'ev¹ and Benjamin Geiger^1,

¹ Department of Molecular Cell Biology, Weizmann Institute of Science Rehovot 76100 Israel
² Sigma Aldrich Israel, Park Rabin, Rehovot 76100, Israel
^* Present address: The Department of Ornamental Horticulture, The Volcani Center, Beit-Dagan, P.O.B. 6, 50250 Israel

View larger version (24K): [in a new window]   Fig. 1. Preparation and characterization of the specific anti-phosphorylated-ß-catenin antibody. (A) A schematic representation of the ß-catenin molecule and the doubly phosphorylated peptide corresponding to its N-terminus that was used to prepare the antibody. Note the homology between ß-catenin and plakoglobin at this region. (B) An ELISA assay for determining the specificity of the antibody. Binding of the antibody to the doubly phosphorylated peptide was detected by a peroxidasebased reaction in the absence of a competitor (first lane on the left) or in the presence of increasing amounts (12.5 25, 50 and 100 µg/ml) of the indicated peptide competitors.

View larger version (28K): [in a new window]   Fig. 2. The anti pß-catenin antibody recognizes wild-type ß-catenin but not mutant ß-catenin or plakoglobin. (A) HA-tagged wild-type ß-catenin or mutant S33Y and S37A-ß-catenin were transfected into 293T cells and analyzed by western blotting with either anti-HA or pß-catenin antibodies. (B) 293T cells were transfected with HA-ß-catenin or with VSV-ß-catenin and HA-F-ß-TrCP. Immunopecipitation was performed with an anti-HA antibody and western blotting with pß-catenin or general ß-catenin antibodies. (C) 293T cells were treated with 30 mM LiCl overnight or with 25 µM MG132 for 4 hours and analyzed by western blotting for pß-catenin and total ß-catenin content. (D) VSV-tagged plakoglobin or ß-catenin were transfected into 293T cells, and their reactivity with pß-catenin antibody was determined when transfected alone or together with HA-tagged F-ß-TrCP. IP, immunoprecipitation; IB, immunoblot.

View larger version (39K): [in a new window]   Fig. 3. ß-catenin phosphorylated on serines 33 and 37 can undergo dephosphorylation. (A) GFP-ß-catenin was transfected into 293T cells, and after 24 hours the cells were treated with either MG132 (lanes 1-4), MG132 plus LiCl (lanes 5-8) or treated with MG132 and LiCl and then washed and incubated with MG132 only (lanes 9-12). (B) 293T cells were treated with MG132 (lanes 2-5), left untreated (lane 1) or incubated with MG132 and LiCl (lanes 6-9) or treated with MG132 and LiCl and then washed from the LiCl and left with only MG132 (lanes 10-12). Cell extracts in (A) and (B) were analyzed by western blotting with pß-catenin antibody and reprobed with general anti-ß-catenin antibody. (C) Quantitative analysis of the level of phosphorylated ß-catenin is shown in B. The numbers correspond to intensities of the signal with pß-catenin antibody normalized to the signal obtained with the general anti-ß-catenin antibody.

View larger version (41K): [in a new window]   Fig. 4. pß-catenin can form a complex with LEF-1 but does not form a ternary complex with LEF-1 bound to DNA. (A) VSV-ß-catenin was co-transfected with HA- LEF-1 into 293T cells and immunoprecipitated with anti HA antibody as a complex with LEF-1. Cell extracts were analyzed by western blotting with the pß-catenin antibody. The asterisk marks the Ig heavy chain of the antibody. (B) VSV-ß-catenin was transfected into 293T cells and immunoprecipitated with ß-catenin or with pß-catenin antibodies as indicated. (C) ß-catenin—LEF-1—DNA complex formation was analyzed with 6 µg of nuclear extracts from 293T cells transfected with LEF-1 and VSV-ß-catenin that were incubated with 32P-labeled DNA containing the LEF-1-binding site, in the absence or presence of the indicated antibodies. Protein-DNA complexes were separated by native 4% PAGE and visualized by autoradiography. The asterisk marks a non-specific band. IP, immunoprecipitation; IB, immunoblot.

View larger version (60K): [in a new window]   Fig. 5. pß-catenin is hardly ever found in complex with N-cadherin. 293T cells were transfected with ß-catenin together with either N-cadherin or with F-ß-TrCP, which served as a control for complexing with pß-catenin. Cell extracts were immunoprecipitated as indicated and analyzed by western blotting with both the pß-catenin and a general anti-ß-catenin antibodies. The asterisks mark the Ig heavy chain of the antibody.

View larger version (64K): [in a new window]   Fig. 6. Blocking proteasomal degradation leads to nuclear accumulation of ß-catenin. (A) BCAP cells were treated with MG132 for 4 hours and then fixed and stained as indicated. Bar, 10 µm. (B) Cell extracts from the MG132-treated and control BCAP cells were analyzed by western blotting. vin, vinculin; pß-cat, phosphorylated ß-catenin.

View larger version (68K): [in a new window]   Fig. 7. pß-catenin is transiently associated with nascent adherens junctions. (a) MDCK cells were transfected with HA-ß-catenin and after 48 hours fixed and stained as described in the Materials and Methods. Ratio imaging analysis was carried out at six optical sections (0.6 µm apart) and shows exclusive localization of pß-catenin (red) in nuclear speckles and not in adherens junctions. (b) BCAP cells were plated on coverslips for the specified times (A-D) or treated with 4 mM EGTA for 1 hour (E) or washed and allowed to recover for 3 hours in the presence of fresh medium containing Ca2+ (F). The cells were fixed, stained and analyzed by ratio imaging analysis. The colors represent the ratio between pß-catenin (red) and either HA ß-catenin (blue, in the transfected MDCK cells) or total endogenous ß-catenin (blue, in the BCAP cells). The insert in A shows the specific inhibition of the pß-catenin staining in the presence of the double phosphorylated peptide (pep). Bar, 10 µm.

View larger version (30K): [in a new window]   Fig. 8. pß-catenin expression in colon cancer cell lines. HT29, SW480 and HCT116 colon carcinoma cells and 293T cells were either treated (+) with MG132 for 4 hours or left untreated (-). Cell extracts were analyzed by western blotting using both the pß-catenin antibody and a general anti-ß-catenin antibody.

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