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First published online November 8, 2006
doi: 10.1242/10.1242/jcs.03256

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Protein-kinase-C-mediated ß-catenin phosphorylation negatively regulates the Wnt/ß-catenin pathway

¹ PharmcoGenomics Research Center, Inje University, Busan 614-735, Korea
² Department of Biotechnology and Bioengineering, Dong-Eui University, Busan 614-714, Korea
³ Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 305-701, Korea
⁴ Department of Clinical Pharmacology, Busan Paik Hospital, Inje University College of Medicine, Busan 614-735, Korea
⁵ Department of Pharmacology, Inje University College of Medicine, Busan 614-735, Korea
⁶ CGK Co. Ltd, Daejeon 305-701, Korea

View larger version (22K): [in a new window]   Fig. 1. Identification of A23187 as a small-molecule inhibitor of Wnt/ß-catenin signaling. (A) Screening of compounds that inhibit Wnt/ß-catenin signaling. (B) Dose-dependent inhibition of CRT induction plotted as increasing concentrations of A23187. HEK293 reporter and control cells were incubated with A23187 (0.625, 1.25, 2.5 and 5 µM) for 15 hours in the presence or absence of Wnt3a-CM, and luciferase activity was determined. The results shown are the average of three experiments; the bars indicate standard deviations. (C) Cytosolic proteins were prepared from HEK293 reporter cells treated with either vehicle (DMSO) or A23187 (2.5 µM) in the presence or absence of Wnt3a-CM and were then subjected to western blotting with anti-ß-catenin antibody. (D) Cytosolic proteins prepared from HEK293 reporter cells were incubated with vehicle (DMSO) or A23187 (2.5 µM) in the presence or absence of Wnt3a-CM, exposed to MG-132 (20 µM) for 8 hours, and then subjected to western blotting with anti-ß-catenin antibody. In C and D, the blots were reprobed with anti-actin antibody as a loading control.

View larger version (31K): [in a new window]   Fig. 2. A23187 promotes ß-catenin degradation through GSK-3ß-independent and ß-TrCP-dependent mechanism. (A) HEK293 reporter cells were incubated with A23187 (0.625, 1.25, 2.5 and 5 µM) in the presence of 20 mM LiCl. After 15 hours, luciferase activity was determined. (B) Cytosolic proteins were prepared from HEK293 reporter cells treated with either vehicle (DMSO) or A23187 (2.5 µM) in the presence of 20 mM LiCl for 15 hours and then subjected to western blotting with ß-catenin antibody. The blots were reprobed with anti-actin antibody as a loading control. (C) HEK293 cells were co-transfected with the indicated plasmids and then incubated with either the vehicle (DMSO) or A23187 (2.5 µM) for 15 hours. Cytosolic proteins were subjected to western blotting with ß-catenin antibody. The blots were reprobed with anti-GFP antibody as a transfection control. (D) HEK293 reporter cells were co-transfected with the indicated plasmids and then incubated with either the vehicle (DMSO) or A23187 (2.5 µM) for 15 hours and then luciferase activity was measured. In A and D, the results are the average of three experiments, and the bars indicated standard deviation.

View larger version (34K): [in a new window]   Fig. 3. The N-terminus of ß-catenin is required for A23187-mediated ß-catenin degradation. HEK293 cells were transfected with wild-type ß-catenin (A), ß-catenin (B) or ß-catenin (S37A) (C) plasmids, incubated with A23187 [1.25 (+) and 2.5 (++) µM] for 15 hours, and then cytosolic proteins were immunoblotted with anti-ß-catenin antibody. The blots were reprobed with anti-GFP antibody as a transfection control.

View larger version (27K): [in a new window]   Fig. 4. PKC is required for A23187-mediated degradation of ß-catenin. (A) A23187 increases the intracellular Ca2+ concentration. HEK293 reporter cells were incubated with vehicle (DMSO) or A23187 (2.5 µM) in the presence or absence of Wnt3a-CM for 15 hours. After fixation, the cells were stained with Fura-2/AM as described in Materials and Methods and observed at 400x magnification. (B) HEK293 reporter cells were incubated with A23187 (2.5 µM) or CsA [2.5 (+), 5 (++) µM], KN-93 [2.5 (+), 5 (++) µM] and BIM [2.5 (+), 5 (++) µM] for 15 hours in the presence or absence of Wnt3a-CM, and the luciferase activity was determined. The results are shown as the average of three experiments; the bars indicate standard deviations. (C) Cytosolic proteins were prepared from HEK293 reporter cells treated with A23187 (2.5 µM) or BIM (5 µM) in the absence or presence of Wnt3a-CM for western blotting with anti-ß-catenin antibody. The blots were reprobed with anti-actin antibody as a loading control.

View larger version (37K): [in a new window]   Fig. 5. A23187 induces the translocation of PKC to the membrane. (A) Cytosolic and membrane fractions were prepared from HEK293 reporter cells treated with vehicle (DMSO) or A23187 (2.5 µM) in the presence or absence of Wnt3a-CM, for western blotting with anti-PKC antibody. The blots were reprobed with anti-actin or anti-MDR antibodies as fractionation controls. (B) The cellular location of PKC in HEK293 reporter cells was determined by immunofluorescence analysis. After fixation, the cells were stained with anti-PKC antibody and observed at 400x magnification. Arrows indicate PKC translocated to the membrane.

View larger version (34K): [in a new window]   Fig. 6. PKC phosphorylates N-terminal Ser residues of ß-catenin in vitro. GST-ß-catenin (100 ng) was incubated with the indicated amount of purified PKC (A) or BIM (0.5 µM; B), and then the samples were analyzed by western blotting with anti-phospho-p45-ß-catenin and anti-phospho-p33/37-ß-catenin antibodies.

View larger version (36K): [in a new window]   Fig. 7. PKC phosphorylates N-terminal Ser residues of ß-catenin in vivo. (A) HEK293 reporter cells were incubated with A23187 (2.5 µM) and BIM (5 µM) for 15 h in the absence or presence of Wnt3a-CM. Cytosolic fractions were prepared and subjected to western blot analysis with anti-phospho-p45-ß-catenin, anti-phospho-p33/37-ß-catenin or anti-ß-catenin antibody. The same amount of ß-catenin was loaded in each lane. (B) HEK293 cells were transfected with negative control (NC) siRNA (40 µmol) and PKC siRNA (20 and 40 µmol) for 48 hours and then cell lysates were subjected to western blot analysis with anti-PKC, anti-ß-catenin, anti-p45-ß-catenin, anti-p33/37-ß-catenin and anti-actin antibodies. (C) HEK293 reporter cells were transfected with negative control siRNA (40 µmol) and PKC siRNA (40 µmol) for 48 hours and then luciferase activity was measured. The results are the average of three experiments, and the bars indicated standard deviation.

View larger version (13K): [in a new window]   Fig. 8. Inhibition of PKC partially abolished the Wnt5a-mediated CRT inhibition. (A) HEK293 reporter and control cells were co-transfected with Wnt3a (50 ng) and Wnt5a [0.2 µg (+) and 1 µg (++)] plasmids for 48 hours, and then luciferase activity was determined. (B) HEK293 reporter and control cells were co-transfected with Wnt3a (50 ng) and Wnt5a (1 µg) plasmids, incubated with BIM [2.5 µM (+) and 5 µM (++)] for 15 hours, and then luciferase activity was measured. In A and B, the results shown are the average of three experiments; the bars indicated standard deviations.

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