Limits...
Physiological regulation of [beta]-catenin stability by Tcf3 and CK1epsilon.

Lee E, Salic A, Kirschner MW - J. Cell Biol. (2001)

Bottom Line: Tcf3 is a substrate for both glycogen synthase kinase (GSK) 3 and casein kinase (CK) 1epsilon, and phosphorylation of Tcf3 by CKIepsilon stimulates its binding to beta-catenin, an effect reversed by GSK3.Tcf3 synergizes with CK1epsilon to inhibit beta-catenin degradation, whereas CKI-7, an inhibitor of CK1epsilon, reduces the inhibitory effect of Tcf3.Along with evidence that a significant amount of Tcf protein is nonnuclear, these findings suggest that CK1epsilon can modulate wnt signaling in vivo by regulating both the beta-catenin-Tcf3 and the GBP-dsh interfaces.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.

ABSTRACT
The wnt pathway regulates the steady state level of beta-catenin, a transcriptional coactivator for the Tcf3/Lef1 family of DNA binding proteins. We demonstrate that Tcf3 can inhibit beta-catenin turnover via its competition with axin and adenomatous polyposis for beta-catenin binding. A mutant of beta-catenin that cannot bind Tcf3 is degraded faster than the wild-type protein in Xenopus embryos and extracts. A fragment of beta-catenin and a peptide encoding the NH2 terminus of Tcf4 that block the interaction between beta-catenin and Tcf3 stimulate beta-catenin degradation, indicating this interaction normally plays an important role in regulating beta-catenin turnover. Tcf3 is a substrate for both glycogen synthase kinase (GSK) 3 and casein kinase (CK) 1epsilon, and phosphorylation of Tcf3 by CKIepsilon stimulates its binding to beta-catenin, an effect reversed by GSK3. Tcf3 synergizes with CK1epsilon to inhibit beta-catenin degradation, whereas CKI-7, an inhibitor of CK1epsilon, reduces the inhibitory effect of Tcf3. Finally, we provide evidence that CK1epsilon stimulates the binding of dishevelled (dsh) to GSk3 binding protein (GBP) in extracts. Along with evidence that a significant amount of Tcf protein is nonnuclear, these findings suggest that CK1epsilon can modulate wnt signaling in vivo by regulating both the beta-catenin-Tcf3 and the GBP-dsh interfaces.

Show MeSH

Related in: MedlinePlus

Role of CK1ε in mediating dsh activity. (A) dsh acts synergistically with CK1ε in Xenopus embryos to induce axis duplication. Embryos were injected with 50 pg CK1ε, 100 pg dsh, or 50 pg CK1ε plus 100 pg dsh RNA in one ventral blastomere of 4–8-cell stage embryos. (B) Dsh and CK1e act synergistically to inhibit β-catenin degradation. Dsh protein (100 nM) and CK1e protein (500 nM) were added to extracts alone or in combination. (C) GBP cross-linked beads were incubated with radiolabeled dsh and either buffer, 1 μM CK1ε, or 1 μM GSK3 in the presence or absence of Xenopus extracts. CK1ε stimulates the binding of GBP to radiolabeled dsh in Xenopus extracts. No differences were detected when binding was performed in the absence of extracts.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2196183&req=5

fig8: Role of CK1ε in mediating dsh activity. (A) dsh acts synergistically with CK1ε in Xenopus embryos to induce axis duplication. Embryos were injected with 50 pg CK1ε, 100 pg dsh, or 50 pg CK1ε plus 100 pg dsh RNA in one ventral blastomere of 4–8-cell stage embryos. (B) Dsh and CK1e act synergistically to inhibit β-catenin degradation. Dsh protein (100 nM) and CK1e protein (500 nM) were added to extracts alone or in combination. (C) GBP cross-linked beads were incubated with radiolabeled dsh and either buffer, 1 μM CK1ε, or 1 μM GSK3 in the presence or absence of Xenopus extracts. CK1ε stimulates the binding of GBP to radiolabeled dsh in Xenopus extracts. No differences were detected when binding was performed in the absence of extracts.

Mentions: Given the fact that CK1ε has been shown to bind axin (Sakanaka et al., 1999) and dsh (Peters et al., 1999), it appears that CK1ε like GSK3 simultaneously affects many components of the wnt pathway. Dsh acts synergistically with CK1ε in Xenopus embryos (Fig. 8 A). Embryos injected ventrally at the 4–8-cell stage with low doses of Dsh (100 pg) or CK1ε (50 pg) RNA develop normally (100%, n = 30), whereas injection of embryos with both Dsh and CK1ε resulted in axis duplication (68%, n = 50). Dsh acts synergistically with CK1ε to inhibit β-catenin degradation in Xenopus extracts (Fig. 8 B), consistent with the in vivo results. Preliminary experiments indicate that both GSK3 and CK1ε can bind the PDZ domain of dsh (unpublished data). Since GBP binds the PDZ domain of dsh, we tested whether CK1ε or GSK3 can alter the affinity of dsh for GBP. CK1ε stimulates the binding of radiolabeled dsh to GBP in Xenopus extracts four- to fivefold greater than in buffer- or GSK3-treated extracts (Fig. 8 C). This result indicates that in addition to promoting the association between Tcf3 and β-catenin, CK1ε stimulates the binding of GBP to dsh. Interestingly, in the absence of Xenopus extracts CK1ε has no effect on binding of dsh to GBP, suggesting that extracts contain an activity that mediates the effect of CK1ε on the dsh–GBP interaction.


Physiological regulation of [beta]-catenin stability by Tcf3 and CK1epsilon.

Lee E, Salic A, Kirschner MW - J. Cell Biol. (2001)

Role of CK1ε in mediating dsh activity. (A) dsh acts synergistically with CK1ε in Xenopus embryos to induce axis duplication. Embryos were injected with 50 pg CK1ε, 100 pg dsh, or 50 pg CK1ε plus 100 pg dsh RNA in one ventral blastomere of 4–8-cell stage embryos. (B) Dsh and CK1e act synergistically to inhibit β-catenin degradation. Dsh protein (100 nM) and CK1e protein (500 nM) were added to extracts alone or in combination. (C) GBP cross-linked beads were incubated with radiolabeled dsh and either buffer, 1 μM CK1ε, or 1 μM GSK3 in the presence or absence of Xenopus extracts. CK1ε stimulates the binding of GBP to radiolabeled dsh in Xenopus extracts. No differences were detected when binding was performed in the absence of extracts.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196183&req=5

fig8: Role of CK1ε in mediating dsh activity. (A) dsh acts synergistically with CK1ε in Xenopus embryos to induce axis duplication. Embryos were injected with 50 pg CK1ε, 100 pg dsh, or 50 pg CK1ε plus 100 pg dsh RNA in one ventral blastomere of 4–8-cell stage embryos. (B) Dsh and CK1e act synergistically to inhibit β-catenin degradation. Dsh protein (100 nM) and CK1e protein (500 nM) were added to extracts alone or in combination. (C) GBP cross-linked beads were incubated with radiolabeled dsh and either buffer, 1 μM CK1ε, or 1 μM GSK3 in the presence or absence of Xenopus extracts. CK1ε stimulates the binding of GBP to radiolabeled dsh in Xenopus extracts. No differences were detected when binding was performed in the absence of extracts.
Mentions: Given the fact that CK1ε has been shown to bind axin (Sakanaka et al., 1999) and dsh (Peters et al., 1999), it appears that CK1ε like GSK3 simultaneously affects many components of the wnt pathway. Dsh acts synergistically with CK1ε in Xenopus embryos (Fig. 8 A). Embryos injected ventrally at the 4–8-cell stage with low doses of Dsh (100 pg) or CK1ε (50 pg) RNA develop normally (100%, n = 30), whereas injection of embryos with both Dsh and CK1ε resulted in axis duplication (68%, n = 50). Dsh acts synergistically with CK1ε to inhibit β-catenin degradation in Xenopus extracts (Fig. 8 B), consistent with the in vivo results. Preliminary experiments indicate that both GSK3 and CK1ε can bind the PDZ domain of dsh (unpublished data). Since GBP binds the PDZ domain of dsh, we tested whether CK1ε or GSK3 can alter the affinity of dsh for GBP. CK1ε stimulates the binding of radiolabeled dsh to GBP in Xenopus extracts four- to fivefold greater than in buffer- or GSK3-treated extracts (Fig. 8 C). This result indicates that in addition to promoting the association between Tcf3 and β-catenin, CK1ε stimulates the binding of GBP to dsh. Interestingly, in the absence of Xenopus extracts CK1ε has no effect on binding of dsh to GBP, suggesting that extracts contain an activity that mediates the effect of CK1ε on the dsh–GBP interaction.

Bottom Line: Tcf3 is a substrate for both glycogen synthase kinase (GSK) 3 and casein kinase (CK) 1epsilon, and phosphorylation of Tcf3 by CKIepsilon stimulates its binding to beta-catenin, an effect reversed by GSK3.Tcf3 synergizes with CK1epsilon to inhibit beta-catenin degradation, whereas CKI-7, an inhibitor of CK1epsilon, reduces the inhibitory effect of Tcf3.Along with evidence that a significant amount of Tcf protein is nonnuclear, these findings suggest that CK1epsilon can modulate wnt signaling in vivo by regulating both the beta-catenin-Tcf3 and the GBP-dsh interfaces.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.

ABSTRACT
The wnt pathway regulates the steady state level of beta-catenin, a transcriptional coactivator for the Tcf3/Lef1 family of DNA binding proteins. We demonstrate that Tcf3 can inhibit beta-catenin turnover via its competition with axin and adenomatous polyposis for beta-catenin binding. A mutant of beta-catenin that cannot bind Tcf3 is degraded faster than the wild-type protein in Xenopus embryos and extracts. A fragment of beta-catenin and a peptide encoding the NH2 terminus of Tcf4 that block the interaction between beta-catenin and Tcf3 stimulate beta-catenin degradation, indicating this interaction normally plays an important role in regulating beta-catenin turnover. Tcf3 is a substrate for both glycogen synthase kinase (GSK) 3 and casein kinase (CK) 1epsilon, and phosphorylation of Tcf3 by CKIepsilon stimulates its binding to beta-catenin, an effect reversed by GSK3. Tcf3 synergizes with CK1epsilon to inhibit beta-catenin degradation, whereas CKI-7, an inhibitor of CK1epsilon, reduces the inhibitory effect of Tcf3. Finally, we provide evidence that CK1epsilon stimulates the binding of dishevelled (dsh) to GSk3 binding protein (GBP) in extracts. Along with evidence that a significant amount of Tcf protein is nonnuclear, these findings suggest that CK1epsilon can modulate wnt signaling in vivo by regulating both the beta-catenin-Tcf3 and the GBP-dsh interfaces.

Show MeSH
Related in: MedlinePlus