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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.

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Tcf3 blocks β-catenin degradation in extracts and phosphorylation in vitro. (A) Translated Tcf3 but not ΔNTcf3 mRNA inhibits β-catenin degradation in extracts. (B) Purified Tcf3 protein (1 μM) blocks β-catenin degradation. This effect was not reversed by 1 μM GSK3. Purified ΔNTcf3 (1 μM) does not block β-catenin degradation. (C) Inhibition of β-catenin degradation by lower (100 nM) Tcf3 levels can be partially rescued by GSK3; higher Tcf3 (300 nM) levels cannot be rescued by even a large GSK3 excess. (D) Tcf3 inhibits the phosphorylation of β-catenin by GSK3 and axin in a purified system. In the same reaction, axin phosphorylation by GSK3 is not affected by Tcf3.
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fig1: Tcf3 blocks β-catenin degradation in extracts and phosphorylation in vitro. (A) Translated Tcf3 but not ΔNTcf3 mRNA inhibits β-catenin degradation in extracts. (B) Purified Tcf3 protein (1 μM) blocks β-catenin degradation. This effect was not reversed by 1 μM GSK3. Purified ΔNTcf3 (1 μM) does not block β-catenin degradation. (C) Inhibition of β-catenin degradation by lower (100 nM) Tcf3 levels can be partially rescued by GSK3; higher Tcf3 (300 nM) levels cannot be rescued by even a large GSK3 excess. (D) Tcf3 inhibits the phosphorylation of β-catenin by GSK3 and axin in a purified system. In the same reaction, axin phosphorylation by GSK3 is not affected by Tcf3.

Mentions: To determine if Tcf3 can influence the rate of β-catenin degradation, we either translated Tcf3 mRNA in Xenopus extracts (Fig. 1 A) or added purified recombinant Tcf3 to the extracts (Fig. 1 B). In both cases, Tcf3 inhibited β-catenin degradation. An IC50 of 30 nM was determined by careful titration of Tcf3 (1 nM to 1 μM) into Xenopus extracts (unpublished data). As a test for the specificity of this interaction, we used an NH2-terminal deletion mutant of Tcf3, ΔNTcf3, which cannot bind β-catenin in vitro (Molenaar et al., 1996). When ΔNTcf3 mRNA was translated in extracts, it had no effect on β-catenin degradation (Fig. 1 A); similarly, ΔNTcf3 purified protein (Fig. 1 B) had no effect. Thus, the inhibitory effect of Tcf3 on β-catenin degradation is dependent on its ability to interact with β-catenin through the NH2-terminal domain. 1 μM of purified GSK3 (Fig. 1 B) did not reverse the inhibitory effect of Tcf3 (1 μM) on β-catenin degradation, suggesting that the effect of Tcf3 was not due to titration of GSK3. A more careful examination showed that excess GSK3 could partially rescue the inhibition of β-catenin degradation by lower levels of Tcf3 (100 nM); however, even a large excess of GSK3 did not reverse the effect of 300 nM added Tcf3 (Fig. 1C).


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

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

Tcf3 blocks β-catenin degradation in extracts and phosphorylation in vitro. (A) Translated Tcf3 but not ΔNTcf3 mRNA inhibits β-catenin degradation in extracts. (B) Purified Tcf3 protein (1 μM) blocks β-catenin degradation. This effect was not reversed by 1 μM GSK3. Purified ΔNTcf3 (1 μM) does not block β-catenin degradation. (C) Inhibition of β-catenin degradation by lower (100 nM) Tcf3 levels can be partially rescued by GSK3; higher Tcf3 (300 nM) levels cannot be rescued by even a large GSK3 excess. (D) Tcf3 inhibits the phosphorylation of β-catenin by GSK3 and axin in a purified system. In the same reaction, axin phosphorylation by GSK3 is not affected by Tcf3.
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Related In: Results  -  Collection

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

fig1: Tcf3 blocks β-catenin degradation in extracts and phosphorylation in vitro. (A) Translated Tcf3 but not ΔNTcf3 mRNA inhibits β-catenin degradation in extracts. (B) Purified Tcf3 protein (1 μM) blocks β-catenin degradation. This effect was not reversed by 1 μM GSK3. Purified ΔNTcf3 (1 μM) does not block β-catenin degradation. (C) Inhibition of β-catenin degradation by lower (100 nM) Tcf3 levels can be partially rescued by GSK3; higher Tcf3 (300 nM) levels cannot be rescued by even a large GSK3 excess. (D) Tcf3 inhibits the phosphorylation of β-catenin by GSK3 and axin in a purified system. In the same reaction, axin phosphorylation by GSK3 is not affected by Tcf3.
Mentions: To determine if Tcf3 can influence the rate of β-catenin degradation, we either translated Tcf3 mRNA in Xenopus extracts (Fig. 1 A) or added purified recombinant Tcf3 to the extracts (Fig. 1 B). In both cases, Tcf3 inhibited β-catenin degradation. An IC50 of 30 nM was determined by careful titration of Tcf3 (1 nM to 1 μM) into Xenopus extracts (unpublished data). As a test for the specificity of this interaction, we used an NH2-terminal deletion mutant of Tcf3, ΔNTcf3, which cannot bind β-catenin in vitro (Molenaar et al., 1996). When ΔNTcf3 mRNA was translated in extracts, it had no effect on β-catenin degradation (Fig. 1 A); similarly, ΔNTcf3 purified protein (Fig. 1 B) had no effect. Thus, the inhibitory effect of Tcf3 on β-catenin degradation is dependent on its ability to interact with β-catenin through the NH2-terminal domain. 1 μM of purified GSK3 (Fig. 1 B) did not reverse the inhibitory effect of Tcf3 (1 μM) on β-catenin degradation, suggesting that the effect of Tcf3 was not due to titration of GSK3. A more careful examination showed that excess GSK3 could partially rescue the inhibition of β-catenin degradation by lower levels of Tcf3 (100 nM); however, even a large excess of GSK3 did not reverse the effect of 300 nM added Tcf3 (Fig. 1C).

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