To determine whether DF3/MUC1 associates with GSK3β, we subjected MAb DF3 (anti-MUC1) immunoprecipitates to immunoblotting with anti-GSK3β. The results demonstrated coprecipitation of GSK3β and MUC1 (Fig. A). In the reciprocal experiment, analysis of anti-GSK3β immunoprecipitates by immunoblotting with the anti-MUC1 antibody confirmed association of MUC1 and GSK3β (Fig. A). To assess whether binding is direct, purified GSK3β was incubated with a GST fusion protein that contains the MUC1 CD (GST-MUC1/CD) (61
). The adsorbate was subjected to immunoblot analysis with anti-GSK3β. The finding that GSK3β binds to GST-MUC1/CD but not to GST alone supported a direct interaction (Fig. B).
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 (more ...)
To identify the site in MUC1 that binds to GSK3β, His-tagged proteins were prepared from the N-terminal (N-MUC1/CD) and C-terminal (C-MUC1/CD) regions of the CD (Fig. A and B, upper panel). Purified GSK3β was incubated with full-length His-MUC1/CD and the two fragments. Immunoprecipitation with anti-GSK3β and analysis of the precipitates with anti-MUC1/CD demonstrated binding of GSK3β with MUC1/CD and C-MUC1/CD but not with the N-MUC1/CD fragment (Fig. B, lower panel). Previous studies have demonstrated that GSK3β phosphorylates an SXXXS site in the APC protein (45
). Since a similar (STDRS) site is present in MUC1/CD, we synthesized an STDRSPYE peptide as a potential competitor for interactions between GSK3β and MUC1. The results demonstrate that preincubation of GSK3β with STDRSPYE inhibits the binding of GSK3β and MUC1/CD (Fig. C). By contrast, there was little effect on this interaction when a control MNRRGSIK peptide was used (Fig. C, upper panel). The STDRSPYE peptide, but not the control peptide, also blocked binding of GSK3β to C-MUC1/CD (Fig. C, lower panel). These findings indicated that GSK3β interacts with the STDRSPYE site in MUC1.
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 (more ...)
To determine whether MUC1/CD is a substrate for GSK3β, we incubated the N-MUC1/CD and C-MUC1/CD fragments with purified GSK3β and [γ-32
P]ATP. Analysis of the reaction products by SDS-PAGE and autoradiography demonstrated phosphorylation of only C-MUC1/CD (data not shown). Previous work has shown that SP sites are substrates for GSK3β phosphorylation (21
). A single SP site in C-MUC1/CD is located in the STDRSPYE domain. Mutation of this domain in MUC1/CD to STDRAPYE [designated MUC1/CD(A)] (Fig. A) abrogated GSK3β-mediated phosphorylation of MUC1/CD (Fig. B). By contrast, mutation to an ATDRSPYE sequence [MUC1/CD(B)] (Fig. A) had little effect on phosphorylation (Fig. B). As expected, the ATDRAPYE double mutant [MUC1/CD(C)] (Fig. A) also failed to serve as a substrate for GSK3β (Fig. B). To assess whether binding of GSK3β to MUC1/CD is affected by the S→A mutations, we incubated GSK3β with wild-type MUC1/CD and the three mutants. Analysis of anti-GSK3β immunoprecipitates by immunoblotting with anti-MUC1/CD demonstrated that the S→A mutations have little if any effect on GSK3β binding (Fig. C). These findings indicate that GSK3β phosphorylates serine in the TDRSPYE domain of MUC1/CD.
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 (more ...)
Previous studies have demonstrated that phosphorylation of APC by GSK3β enhances binding of β-catenin to APC (45
). To assess the effects of GSK3β-mediated phosphorylation of MUC1/CD, we incubated MUC1/CD with GSK3β in the presence and absence of ATP. After phosphorylation of MUC1/CD, GST or GST–β-catenin was added for a 1-h incubation at 4°C. Proteins precipitated with glutathione-Sepharose 4B beads were subjected to immunoblot analysis with anti-MUC1/CD. There was no apparent binding of unphosphorylated or phosphorylated MUC1/CD to GST (Fig. ). By contrast, incubation of MUC1/CD with GST–β-catenin demonstrated that binding of β-catenin is inhibited by GSK3β-mediated phosphorylation of MUC1/CD (Fig. , upper panel). Similar studies with the MUC1/CD(A) mutant also demonstrated decreased binding of β-catenin compared to that of wild-type MUC1/CD. The extent of β-catenin binding to MUC1/CD(A) was unaffected by prior incubation of MUC1/CD(A) with GSK3β and ATP (Fig. , upper panel), consistent with the finding that MUC1/CD(A) is not a substrate for GSK3β. As a control, analysis of adsorbates to the glutathione beads with anti-β-catenin demonstrated that equal amounts of GST–β-catenin were precipitated from the various reaction mixtures (Fig. , lower panel). These findings indicate that modification of the serine in TDRSPYE by phosphorylation or mutation to alanine decreases in vitro binding of MUC1 to β-catenin.
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. (more ...)
To determine whether GSK3β regulates the interaction between MUC1 and β-catenin in vivo, transfection studies were performed in 293 and HeLa cells. 293 cells, in contrast to HeLa cells, have undetectable levels of MUC1 (Fig. A). After transfection of vectors expressing MUC1 and GSK3β, 293 cells were subjected to immunoprecipitation with anti-MUC1 and the precipitates were analyzed for binding of MUC1 to β-catenin. The results demonstrate that GSK3β decreased the interaction between MUC1 and β-catenin compared to control cells transfected with MUC1 alone (Fig. B). By contrast, expression of MUC1 with the kinase-inactive GSK3β(KI) had little effect on the interaction with β-catenin (Fig. B). In studies with the MUC1-positive HeLa cells, transfections were performed with vectors expressing the kinase-active and -inactive GSK3βs. Analysis of anti-MUC1 immunoprecipitates from HeLa cells transfected with the kinase-active GSK3β demonstrated a decrease in the interaction of MUC1 and β-catenin compared to that seen in cells transfected with the empty vector (Fig. C). Moreover, expression of the kinase-inactive GSK3β(KI) had little effect on the interaction of MUC1 and β-catenin (Fig. C). These findings support a model in which GSK3β downregulates binding of MUC1 and β-catenin.
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 (more ...)
To assess whether the interaction of MUC1 and GSK3β affects β-catenin levels, 293 cells were transfected with MUC1 in the absence and presence of GSK3β. Analysis of whole-cell lysates demonstrated little if any change in the total pool of β-catenin in cells that overexpress MUC1 (Fig. A). Similar results were obtained with cells that overexpress both MUC1 and GSK3β (Fig. A). Because these findings do not exclude the possibility that β-catenin is redistributed intracellularly, cytoplasmic and nuclear fractions of the transfectants were analyzed for β-catenin levels. The results demonstrate that overexpression of MUC1 with or without GSK3β has little if any effect on the distribution of β-catenin in the cytoplasm and nucleus (Fig. A). Similar findings were obtained with MUC1-positive HeLa cells transfected to express GSK3β or GSK3β(KI) (Fig. B and data not shown). These findings suggest that overexpression of MUC1 and GSK3β is not associated with redistribution or degradation of β-catenin.
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) (more ...)
β-Catenin binds to the Tcf/LEF family of transcription factors (3
). The functional interaction between β-catenin and Tcf has been assessed by activation of reporter constructs containing the Tcf motif (18
). To investigate whether MUC1 expression affects the transactivation function of β-catenin, 293 cells were transfected with pcDNA3/MUC1 and the β-catenin–Tcf luciferase reporter construct (pTOPFLASH) (18
). As a control, transfections were performed with a similar reporter containing a mutant or nonfunctional Tcf motif (pFOPFLASH) (18
). Transcriptional activity of the pTOPFLASH reporter in 293 cells was approximately 5 to 10 times that of pFOPFLASH (data not shown). Cotransfection with pcDNA3/MUC1 had no apparent effect on pTOPFLASH transcription (Fig. A). The constitutive transcriptional activity of the Tcf reporter gene in 293 cells contrasted with the inactivity of this construct in HeLa cells (data not shown). Therefore, to confirm the findings in 293 cells, we studied the effects of MUC1 expression in SW480 cells, which exhibit a high basal level of transcription of pTOPFLASH (18
). The results demonstrate that transfection of pcDNA3/MUC1 alone or with pcDNA3/GSK3β has no apparent effect on pTOPFLASH activity (Fig. B). These findings indicate that the binding of β-catenin to MUC1 and the regulation of this interaction by GSK3β are involved in functions other than transcriptional activation or repression of the β-catenin–Tcf complex.
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 (more ...)
β-Catenin also interacts with the cell adhesion molecule E-cadherin (14
). The N-terminal domain of β-catenin interacts with α-catenin and thereby links the E-cadherin–β-catenin complex to the cytoskeleton (14
). To determine whether MUC1 influences the interaction between E-cadherin and β-catenin, 293 cells were transfected with pcDNA3/MUC1 and anti-E-cadherin immunoprecipitates were assayed for β-catenin. The results show that MUC1 expression decreases binding of β-catenin to E-cadherin (Fig. A). By contrast, coexpression of MUC1 and GSK3β resulted in restoration of the E-cadherin–β-catenin interaction (Fig. A). The effects of GSK3β were dependent on its kinase function, since coexpression of MUC1 and GSK3β(KI) was associated with a decrease in binding of β-catenin to E-cadherin (Fig. A). Other studies were performed in MUC1-positive HeLa cells by transfecting pcDNA3/GSK3β or pcDNA3/GSK3β(KI). Transfection of GSK3β, which decreases binding of β-catenin to MUC1 (Fig. C), increased the association of β-catenin with E-cadherin (Fig. B). As a control, transfection of GSK3β(KI) had no apparent effect on binding of β-catenin and E-cadherin (Fig. B). These results collectively demonstrate that the interaction of GSK3β and MUC1 decreases binding of β-catenin to MUC1 and stimulates the association of β-catenin and E-cadherin.
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) (more ...)