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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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Effects of Tcf3 on β-catenin mutants. (A) Degradation of both β-catenin and β-cateninΔC2 is inhibited by 1 μM dsh, but only β-catenin is inhibited by 1 μM Tcf3. (B) Graphical representation of densitometry measurements of the autoradiogram in A shows the faster degradation rate of β-cateninΔC2 compared with β-catenin. (C) Both β-catenin and β-cateninΔC2 bind to axin, but only β-catenin binds to xTcf3 in vitro. (D) MBP-cat449/645 inhibits by >95% the binding of [35S]methionine-labeled β-catenin to Tcf3 in vitro (ii). MBP-cat449/645 (2 μM) had no effect on the binding of axin to β-catenin beads (i) and a moderate effect on the binding of β-catenin to APC beads (iii). Binding of radiolabeled β-catenin and axin to control beads was negligible. (E) Degradation of radiolabeled β-catenin in Xenopus extracts is stimulated by MBP-cat449/645 (200 nM). (F) β-catenin–luciferase is degraded more rapidly in embryos when coinjected with MBP-cat449/645. β-catenin–luciferase (4 ng) protein was injected into 2-cell stage Xenopus embryos with or without MBP-cat449/645 (4 ng). At the indicated times, embryos were processed for luciferase assays.
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fig3: Effects of Tcf3 on β-catenin mutants. (A) Degradation of both β-catenin and β-cateninΔC2 is inhibited by 1 μM dsh, but only β-catenin is inhibited by 1 μM Tcf3. (B) Graphical representation of densitometry measurements of the autoradiogram in A shows the faster degradation rate of β-cateninΔC2 compared with β-catenin. (C) Both β-catenin and β-cateninΔC2 bind to axin, but only β-catenin binds to xTcf3 in vitro. (D) MBP-cat449/645 inhibits by >95% the binding of [35S]methionine-labeled β-catenin to Tcf3 in vitro (ii). MBP-cat449/645 (2 μM) had no effect on the binding of axin to β-catenin beads (i) and a moderate effect on the binding of β-catenin to APC beads (iii). Binding of radiolabeled β-catenin and axin to control beads was negligible. (E) Degradation of radiolabeled β-catenin in Xenopus extracts is stimulated by MBP-cat449/645 (200 nM). (F) β-catenin–luciferase is degraded more rapidly in embryos when coinjected with MBP-cat449/645. β-catenin–luciferase (4 ng) protein was injected into 2-cell stage Xenopus embryos with or without MBP-cat449/645 (4 ng). At the indicated times, embryos were processed for luciferase assays.

Mentions: The degradation and binding data suggest that the interaction between β-catenin and Tcf3 could play an important role in vivo in regulating the degradation kinetics of β-catenin. To test if this is the case in vivo, we set out to generate a mutant of β-catenin that is unable to interact with Tcf3. β-catenin COOH-terminal truncation mutants are shown schematically in Fig. 2 E. One would predict that a mutant of β-catenin that cannot interact with Tcf3 would be unresponsive to added Tcf3 and would be degraded at a faster rate than wild-type β-catenin due to its inability to interact with endogenous Tcfs. Constructs were first tested in our degradation assay in Xenopus extracts. Full-length β-catenin and β-cateninΔC2 were degraded similarly (Fig. 2 F); β-cateninΔC1 also behaved identically to the full-length protein (unpublished data). Degradation of full-length β-catenin and β-cateninΔC2 was stimulated by axin (Fig. 2, F and G) and inhibited by lithium (unpublished data), dishevelled (Fig. 3 A), and APCm3 (unpublished data). Mutating the GSK3 phosphorylation sites (serine to alanine) in the NH2-terminal region of β-cateninΔC1 and β-cateninΔC2 stabilized these proteins against degradation in extracts (unpublished data), further demonstrating that the stability of β-cateninΔC1 and β-cateninΔC2 is regulated similarly to wild-type β-catenin. In contrast to β-cateninΔC1 and β-cateninΔC2, β-cateninΔC3 was completely stable (Fig. 2 F), even when 20 nM axin was added to extracts (which normally accelerates the rate of wild-type β-catenin degradation greater than fourfold) (Salic et al., 2000). The stability of β-cateninΔC3 is likely due to its inability to bind axin (unpublished data), an interaction absolutely required for β-catenin degradation (Behrens et al., 1998; Hart et al., 1998; Ikeda et al., 1998; Itoh et al., 1998). Interestingly, degradation of β-cateninΔC2 is not inhibited by up to 1 μM Tcf3 (Fig. 3 A), consistent with its inability to bind Tcf3 (Fig. 3 C). β-cateninΔC2 still binds axin (Fig. 3 C). As with β-catenin, radiolabeled β-cateninΔC2 binds APC in vitro (Fig. 2 H) and can be immunoprecipitated from Xenopus extracts using anti-APC antibody beads (unpublished data). However, unlike full-length β-catenin, this interaction is not blocked by 1 μM Tcf3 added to the extracts (unpublished data). When compared with wild-type β-catenin, β-cateninΔC2 has a significantly faster degradation rate (Fig. 3, A and B), suggesting that endogenous Tcf3 regulates the rate of β-catenin turnover.


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

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

Effects of Tcf3 on β-catenin mutants. (A) Degradation of both β-catenin and β-cateninΔC2 is inhibited by 1 μM dsh, but only β-catenin is inhibited by 1 μM Tcf3. (B) Graphical representation of densitometry measurements of the autoradiogram in A shows the faster degradation rate of β-cateninΔC2 compared with β-catenin. (C) Both β-catenin and β-cateninΔC2 bind to axin, but only β-catenin binds to xTcf3 in vitro. (D) MBP-cat449/645 inhibits by >95% the binding of [35S]methionine-labeled β-catenin to Tcf3 in vitro (ii). MBP-cat449/645 (2 μM) had no effect on the binding of axin to β-catenin beads (i) and a moderate effect on the binding of β-catenin to APC beads (iii). Binding of radiolabeled β-catenin and axin to control beads was negligible. (E) Degradation of radiolabeled β-catenin in Xenopus extracts is stimulated by MBP-cat449/645 (200 nM). (F) β-catenin–luciferase is degraded more rapidly in embryos when coinjected with MBP-cat449/645. β-catenin–luciferase (4 ng) protein was injected into 2-cell stage Xenopus embryos with or without MBP-cat449/645 (4 ng). At the indicated times, embryos were processed for luciferase assays.
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Related In: Results  -  Collection

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fig3: Effects of Tcf3 on β-catenin mutants. (A) Degradation of both β-catenin and β-cateninΔC2 is inhibited by 1 μM dsh, but only β-catenin is inhibited by 1 μM Tcf3. (B) Graphical representation of densitometry measurements of the autoradiogram in A shows the faster degradation rate of β-cateninΔC2 compared with β-catenin. (C) Both β-catenin and β-cateninΔC2 bind to axin, but only β-catenin binds to xTcf3 in vitro. (D) MBP-cat449/645 inhibits by >95% the binding of [35S]methionine-labeled β-catenin to Tcf3 in vitro (ii). MBP-cat449/645 (2 μM) had no effect on the binding of axin to β-catenin beads (i) and a moderate effect on the binding of β-catenin to APC beads (iii). Binding of radiolabeled β-catenin and axin to control beads was negligible. (E) Degradation of radiolabeled β-catenin in Xenopus extracts is stimulated by MBP-cat449/645 (200 nM). (F) β-catenin–luciferase is degraded more rapidly in embryos when coinjected with MBP-cat449/645. β-catenin–luciferase (4 ng) protein was injected into 2-cell stage Xenopus embryos with or without MBP-cat449/645 (4 ng). At the indicated times, embryos were processed for luciferase assays.
Mentions: The degradation and binding data suggest that the interaction between β-catenin and Tcf3 could play an important role in vivo in regulating the degradation kinetics of β-catenin. To test if this is the case in vivo, we set out to generate a mutant of β-catenin that is unable to interact with Tcf3. β-catenin COOH-terminal truncation mutants are shown schematically in Fig. 2 E. One would predict that a mutant of β-catenin that cannot interact with Tcf3 would be unresponsive to added Tcf3 and would be degraded at a faster rate than wild-type β-catenin due to its inability to interact with endogenous Tcfs. Constructs were first tested in our degradation assay in Xenopus extracts. Full-length β-catenin and β-cateninΔC2 were degraded similarly (Fig. 2 F); β-cateninΔC1 also behaved identically to the full-length protein (unpublished data). Degradation of full-length β-catenin and β-cateninΔC2 was stimulated by axin (Fig. 2, F and G) and inhibited by lithium (unpublished data), dishevelled (Fig. 3 A), and APCm3 (unpublished data). Mutating the GSK3 phosphorylation sites (serine to alanine) in the NH2-terminal region of β-cateninΔC1 and β-cateninΔC2 stabilized these proteins against degradation in extracts (unpublished data), further demonstrating that the stability of β-cateninΔC1 and β-cateninΔC2 is regulated similarly to wild-type β-catenin. In contrast to β-cateninΔC1 and β-cateninΔC2, β-cateninΔC3 was completely stable (Fig. 2 F), even when 20 nM axin was added to extracts (which normally accelerates the rate of wild-type β-catenin degradation greater than fourfold) (Salic et al., 2000). The stability of β-cateninΔC3 is likely due to its inability to bind axin (unpublished data), an interaction absolutely required for β-catenin degradation (Behrens et al., 1998; Hart et al., 1998; Ikeda et al., 1998; Itoh et al., 1998). Interestingly, degradation of β-cateninΔC2 is not inhibited by up to 1 μM Tcf3 (Fig. 3 A), consistent with its inability to bind Tcf3 (Fig. 3 C). β-cateninΔC2 still binds axin (Fig. 3 C). As with β-catenin, radiolabeled β-cateninΔC2 binds APC in vitro (Fig. 2 H) and can be immunoprecipitated from Xenopus extracts using anti-APC antibody beads (unpublished data). However, unlike full-length β-catenin, this interaction is not blocked by 1 μM Tcf3 added to the extracts (unpublished data). When compared with wild-type β-catenin, β-cateninΔC2 has a significantly faster degradation rate (Fig. 3, A and B), suggesting that endogenous Tcf3 regulates the rate of β-catenin turnover.

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