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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 competes with axin/APC for β-catenin (cold competitors were present at 1 μM in all experiments). (A) Binding of β-catenin to axin beads is inhibited by Tcf3 but not by ΔNTcf3. In contrast, binding of the β-catenin ΔC2 mutant to axin is unaffected by Tcf3. (B) Tcf3 blocks the binding of β-catenin to axin beads, whereas APCm3 reverses this effect of Tcf3. (C) APCm3 inhibits the binding of β-catenin to Tcf3 beads. (D) 1 μM his6-TCF3 blocks the interaction of β-catenin with endogenous APC in extracts, whereas his6-ΔNTcf3 has no effect. (E) Scheme of the COOH-terminal β-catenin deletion constructs used to map the fragment of β-catenin responsible for stabilization by Tcf3. (F) Normal and axin-induced degradation of β-catenin, β-cateninΔC2, and β-cateninΔC3 in Xenopus extracts. β-cateninΔC3 is completely stable and does not respond to axin. (G) Axin stimulates the turnover of both β-catenin and β-cateninΔC2 in the same degradation reaction. (H) β-catenin and β-cateninΔC2 both bind to APC and binding is not disrupted by the Tcf4 NH2-terminal peptide or by the cat449/645 fragment (at <2 μM).
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fig2: Tcf3 competes with axin/APC for β-catenin (cold competitors were present at 1 μM in all experiments). (A) Binding of β-catenin to axin beads is inhibited by Tcf3 but not by ΔNTcf3. In contrast, binding of the β-catenin ΔC2 mutant to axin is unaffected by Tcf3. (B) Tcf3 blocks the binding of β-catenin to axin beads, whereas APCm3 reverses this effect of Tcf3. (C) APCm3 inhibits the binding of β-catenin to Tcf3 beads. (D) 1 μM his6-TCF3 blocks the interaction of β-catenin with endogenous APC in extracts, whereas his6-ΔNTcf3 has no effect. (E) Scheme of the COOH-terminal β-catenin deletion constructs used to map the fragment of β-catenin responsible for stabilization by Tcf3. (F) Normal and axin-induced degradation of β-catenin, β-cateninΔC2, and β-cateninΔC3 in Xenopus extracts. β-cateninΔC3 is completely stable and does not respond to axin. (G) Axin stimulates the turnover of both β-catenin and β-cateninΔC2 in the same degradation reaction. (H) β-catenin and β-cateninΔC2 both bind to APC and binding is not disrupted by the Tcf4 NH2-terminal peptide or by the cat449/645 fragment (at <2 μM).

Mentions: Phosphorylation and subsequent degradation of β-catenin requires its association with both APC and axin. Since binding of Tcf3 to β-catenin blocks its degradation, we tested if this is due to blocking binding of β-catenin to axin and/or APC. As shown in Fig. 2 A, purified his-tagged Tcf3 but not his-tagged ΔNTcf3 blocked binding of β-catenin to axin beads. However, Tcf3 had no effect on GSK3 binding to axin (unpublished data). A purified 100-kD fragment of APC (APCm3) that spans the axin and β-catenin binding sites stimulates binding of β-catenin to axin beads (Fig. 2 B). In the presence of 100 nM APCm3, addition of up to 1 μM Tcf3 protein had no detectable effect on the binding of β-catenin to axin beads. Furthermore, binding of β-catenin to Tcf3 beads (Fig. 2 C) is effectively inhibited in the presence of 1 μM APCm3. Consistent with a dominant effect of APC in Xenopus extracts, a majority of the soluble β-catenin is in anti-APC immunoprecipitates (Salic et al., 2000). In extracts, 1 μM of added Tcf3 released radiolabeled β-catenin from APC immunoprecipitates (Fig. 2 D). As a control, 1 μM ΔNTcf3 had no detectable effect on β-catenin binding to endogenous APC. These results indicate that Tcf3 competes with both axin and APC for β-catenin binding. The failure of Tcf3 to block β-catenin binding to axin in the presence of APCm3 highlights the strong effect of APC in driving the degradation of free β-catenin by promoting its binding to axin and in inhibiting competing reactions driven by Tcf3. In contrast, the ability of Tcf3 to compete effectively with endogenous APC for β-catenin probably reflects the fact that most of β-catenin is not in a complex with axin/APC, since cellular axin levels are very low (10–20 picomolar; unpublished data).


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

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

Tcf3 competes with axin/APC for β-catenin (cold competitors were present at 1 μM in all experiments). (A) Binding of β-catenin to axin beads is inhibited by Tcf3 but not by ΔNTcf3. In contrast, binding of the β-catenin ΔC2 mutant to axin is unaffected by Tcf3. (B) Tcf3 blocks the binding of β-catenin to axin beads, whereas APCm3 reverses this effect of Tcf3. (C) APCm3 inhibits the binding of β-catenin to Tcf3 beads. (D) 1 μM his6-TCF3 blocks the interaction of β-catenin with endogenous APC in extracts, whereas his6-ΔNTcf3 has no effect. (E) Scheme of the COOH-terminal β-catenin deletion constructs used to map the fragment of β-catenin responsible for stabilization by Tcf3. (F) Normal and axin-induced degradation of β-catenin, β-cateninΔC2, and β-cateninΔC3 in Xenopus extracts. β-cateninΔC3 is completely stable and does not respond to axin. (G) Axin stimulates the turnover of both β-catenin and β-cateninΔC2 in the same degradation reaction. (H) β-catenin and β-cateninΔC2 both bind to APC and binding is not disrupted by the Tcf4 NH2-terminal peptide or by the cat449/645 fragment (at <2 μM).
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Related In: Results  -  Collection

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fig2: Tcf3 competes with axin/APC for β-catenin (cold competitors were present at 1 μM in all experiments). (A) Binding of β-catenin to axin beads is inhibited by Tcf3 but not by ΔNTcf3. In contrast, binding of the β-catenin ΔC2 mutant to axin is unaffected by Tcf3. (B) Tcf3 blocks the binding of β-catenin to axin beads, whereas APCm3 reverses this effect of Tcf3. (C) APCm3 inhibits the binding of β-catenin to Tcf3 beads. (D) 1 μM his6-TCF3 blocks the interaction of β-catenin with endogenous APC in extracts, whereas his6-ΔNTcf3 has no effect. (E) Scheme of the COOH-terminal β-catenin deletion constructs used to map the fragment of β-catenin responsible for stabilization by Tcf3. (F) Normal and axin-induced degradation of β-catenin, β-cateninΔC2, and β-cateninΔC3 in Xenopus extracts. β-cateninΔC3 is completely stable and does not respond to axin. (G) Axin stimulates the turnover of both β-catenin and β-cateninΔC2 in the same degradation reaction. (H) β-catenin and β-cateninΔC2 both bind to APC and binding is not disrupted by the Tcf4 NH2-terminal peptide or by the cat449/645 fragment (at <2 μM).
Mentions: Phosphorylation and subsequent degradation of β-catenin requires its association with both APC and axin. Since binding of Tcf3 to β-catenin blocks its degradation, we tested if this is due to blocking binding of β-catenin to axin and/or APC. As shown in Fig. 2 A, purified his-tagged Tcf3 but not his-tagged ΔNTcf3 blocked binding of β-catenin to axin beads. However, Tcf3 had no effect on GSK3 binding to axin (unpublished data). A purified 100-kD fragment of APC (APCm3) that spans the axin and β-catenin binding sites stimulates binding of β-catenin to axin beads (Fig. 2 B). In the presence of 100 nM APCm3, addition of up to 1 μM Tcf3 protein had no detectable effect on the binding of β-catenin to axin beads. Furthermore, binding of β-catenin to Tcf3 beads (Fig. 2 C) is effectively inhibited in the presence of 1 μM APCm3. Consistent with a dominant effect of APC in Xenopus extracts, a majority of the soluble β-catenin is in anti-APC immunoprecipitates (Salic et al., 2000). In extracts, 1 μM of added Tcf3 released radiolabeled β-catenin from APC immunoprecipitates (Fig. 2 D). As a control, 1 μM ΔNTcf3 had no detectable effect on β-catenin binding to endogenous APC. These results indicate that Tcf3 competes with both axin and APC for β-catenin binding. The failure of Tcf3 to block β-catenin binding to axin in the presence of APCm3 highlights the strong effect of APC in driving the degradation of free β-catenin by promoting its binding to axin and in inhibiting competing reactions driven by Tcf3. In contrast, the ability of Tcf3 to compete effectively with endogenous APC for β-catenin probably reflects the fact that most of β-catenin is not in a complex with axin/APC, since cellular axin levels are very low (10–20 picomolar; unpublished data).

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