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, 18 (12), 7216-24

Interaction of Glycogen Synthase Kinase 3beta With the DF3/MUC1 Carcinoma-Associated Antigen and Beta-Catenin

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Interaction of Glycogen Synthase Kinase 3beta With the DF3/MUC1 Carcinoma-Associated Antigen and Beta-Catenin

Y Li et al. Mol Cell Biol.

Abstract

The DF3/MUC1 mucin-like glycoprotein is highly overexpressed in human carcinomas. Recent studies have demonstrated that the cytoplasmic domain of MUC1 interacts with beta-catenin. Here we show that MUC1 associates with glycogen synthase kinase 3beta (GSK3beta). GSK3beta binds directly to an STDRSPYE site in MUC1 and phosphorylates the serine adjacent to proline. Phosphorylation of MUC1 by GSK3beta decreases binding of MUC1 to beta-catenin in vitro and in vivo. GSK3beta-mediated phosphorylation of MUC1 had no apparent effect on beta-catenin levels or the transcriptional coactivation function of beta-catenin. The results, however, demonstrate that MUC1 expression decreases binding of beta-catenin to the E-cadherin cell adhesion molecule. Negative regulation of the beta-catenin-MUC1 interaction by GSK3beta is associated with restoration of the complex between beta-catenin and E-cadherin. These findings indicate that GSK3beta decreases the interaction of MUC1 with beta-catenin and that overexpression of MUC1 in the absence of GSK3beta activity inhibits formation of the E-cadherin-beta-catenin complex.

Figures

FIG. 1
FIG. 1
Interaction of MUC1 and GSK3β. (A) Lysates from ZR-75-1 cells were subjected to immunoprecipitation with anti-MUC1 (MAb DF3; upper panel) or anti-GSK3β (lower panel). Mouse IgG was used as a control. The immunoprecipitates were analyzed by immunoblotting with anti-GSK3β (upper panel) or anti-MUC1 (lower panel). (B) GST and GST-MUC1/CD were incubated with purified GSK3β. Proteins precipitated with glutathione-Sepharose 4B beads were separated by SDS-PAGE, transferred to nitrocellulose membranes, and immunoblotted with anti-GSK3β. Purified GSK3β was directly subjected to immunoblot analysis with anti-GSK3β as a control. The positions of molecular size markers are shown on the left of the gels.
FIG. 2
FIG. 2
GSK3β interacts with the STDRSPYE site in MUC1/CD. (A) Amino acid sequences of the MUC1/CD, N-MUC1/CD, and C-MUC1/CD proteins. The 72-amino-acid CD is reflected by numbering from the N terminus of the expressed MUC1/CD protein. The β-catenin binding sequence is boxed, and the GSK3β binding and phosphorylation site is underlined. (B) The purified N-MUC1/CD, full-length MUC1/CD, and C-MUC1/CD proteins were subjected to immunoblotting (IB) with an anti-MUC1/CD antibody (upper panel). Purified N-MUC1/CD, MUC1/CD, and c-MUC1/CD were incubated with purified GSK3β. Complexes immunoprecipitated (IP) with anti-GSK3β were subjected to immunoblotting with anti-MUC1/CD (lower panel). The position of a molecular size standard is shown on the left of both panels. (C) Purified MUC1/CD (upper panel) and C-MUC1/CD (lower panel) were incubated with purified GSK3β in the absence of competing peptide and in the presence of the STDRSPYE peptide or an irrelevant control peptide. Anti-GSK3β immunoprecipitates were analyzed by immunoblotting with anti-MUC1/CD.
FIG. 3
FIG. 3
GSK3β phosphorylates MUC1/CD at the TDRSPYE domain. (A) Wild-type and mutant forms of MUC1/CD. TR, tandem repeat; TM, transmembrane. Numbers (1 to 72) reflect amino acids in the CD. Underlined codons and amino acids are those that differ from the wild type. (B) Purified MUC1/CD proteins were incubated with purified GSK3β and [γ32P]ATP. As a control, MUC1/CD was incubated with [γ32P]ATP and no GSK3β. The reaction products were analyzed by SDS-PAGE and autoradiography (upper panel). Equal loading of the MUC1/CD proteins was assessed by Coomassie blue staining (lower panel). The position of a molecular size standard is shown on the left. (C) Purified MUC1/CD proteins were incubated with purified GSK3β. The proteins were subjected to immunoprecipitation (IP) with anti-GSK3β, and the precipitates were analyzed by immunoblotting (IB) with anti-MUC1/CD. The control lane represents incubation of MUC1/CD and GSK3β, immunoprecipitation with mouse IgG, and immunoblot analysis of the precipitates with anti-MUC1/CD.
FIG. 4
FIG. 4
GSK3β-mediated phosphorylation of the TDRSPYE site in vitro reduces binding of MUC1 to β-catenin. MUC1/CD and MUC1/CD(A) were incubated with (+) or without (−) purified GSK3β and ATP for 15 min at 30°C. The MUC1/CD and MUC1/CD(A) proteins were then incubated with GST or GST–β-catenin for 1 h at 4°C. Proteins precipitated with glutathione-Sepharose 4B beads were subjected to immunoblot (IB) analysis with anti-MUC1 (upper panel) and anti-β-catenin (lower panel). The positions of molecular size standards are shown on the left of the gels.
FIG. 5
FIG. 5
GSK3β downregulates the interaction between MUC1 and β-catenin. (A) Lysates from 293 and HeLa cells were subjected to immunoblot (IB) analysis with anti-MUC1. (B) 293 cells were transiently transfected with pcDNA3 (10 μg), pcDNA3 plus MUC1 (5 μg each), MUC1 plus GSK3β (5 μg each), or MUC1 plus GSK3β(KI) (5 μg each). After 48 h, the cells were harvested and lysates were subjected to immunoprecipitation (IP) with anti-MUC1. The immunoprecipitates were analyzed by immunoblotting with anti-β-catenin. As a control, 293 cell lysate was directly analyzed by immunoblotting with anti-β-catenin (last lane). (C) HeLa cells were transiently transfected with pcDNA3 (10 μg), GSK3β (10 μg), or GSK3β(KI) (10 μg). After 48 h, lysates were prepared from the transfected cells and proteins were immunoprecipitated with anti-MUC1. The immunoprecipitates were analyzed by immunoblotting with anti-β-catenin. HeLa cell lysate was directly analyzed by immunoblotting with anti-β-catenin. The positions of molecular size standards are shown to the left of all panels.
FIG. 6
FIG. 6
Effect of MUC1 and GSK3β on β-catenin levels. (A) 293 cells were transiently transfected with pcDNA3, pcDNA3/MUC1, or MUC1/GSK3β. After 48 h, the cells were harvested and total cell lysates (TCL) were subjected to immunoblot (IB) analysis with anti-β-catenin. The cell lysates were also separated into nuclear (N) and cytoplasmic (C) fractions that were analyzed by immunoblotting with anti-β-catenin. (B) HeLa cells were transfected with pcDNA3, pcDNA3/GSK3β, or pcDNA3/GSK3β(KI). Total cell lysates were analyzed by immunoblotting with anti-β-catenin.
FIG. 7
FIG. 7
Effect of MUC1 on the cotransfection function of β-catenin. 293 (A) and SW480 (B) cells were transiently transfected with 0.3 μg of pTOPFLASH (solid bars) or pFOPFLASH (hatched bars), the indicated amounts of MUC1 vector, 0.3 μg of pCATCONTROL, and pcDNA3 to a total of 2.5 μg of plasmid DNA. After 48 h, the cells were harvested and cell lysates were assayed for luciferase activity. The results of two independent experiments are shown.
FIG. 8
FIG. 8
Regulation of β-catenin–E-cadherin complexes by MUC1 and GSK3β. (A) 293 cells were transfected with pcDNA3 (10 μg), pcDNA3 plus MUC1 (5 μg each), MUC1 plus GSK3β (5 μg each), or MUC1 plus GSK3β(KI) (5 μg each). (B) HeLa cells were transfected with pcDNA3 (10 μg), GSK3β (10 μg), or GSK3β(KI) (10 μg). After 48 h, the transfected cells were harvested and cell lysates subjected to immunoprecipitation (IP) with anti-E-cadherin. The precipitates were analyzed by immunoblotting (IB) with anti-β-catenin. As controls, cell lysates were directly subjected to immunoblot analysis with anti-β-catenin (last lanes).

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