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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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GSK3 and CK1ε bind and phosphorylate Tcf3. (A) Tcf3 purified from Sf9 cells contains lithium-sensitive kinase activity. Recombinant his6-Tcf3 (1μg) was incubated in 10 μl kinase buffer (described in Materials and methods) for 30 min at room temperature either in the presence or absence of 100 mM LiCl, which normally inhibits GSK3 activity. Phosphorylation of Tcf3 is dramatically decreased in the presence of lithium. (B) Tcf3 beads pull down GSK3 from Xenopus extracts. Beads (control-BSA or Tcf3-coupled) were incubated with Xenopus egg extracts, washed, eluted, and analyzed by Western blotting with a monoclonal anti-GSK3 antibody. (C) Both GSK3 and CK1ε bind Tcf3. Radiolabeled in vitro–translated GSK3 and CK1ε bind Tcf3 beads. Binding of radiolabeled GSK3 and CK1ε to Tcf3 beads was abolished by addition of excess cold protein (5 μM of his6-GSK3 and MBP-CK1ε, respectively), demonstrating specificity of the binding reaction. However, excess cold his6-GSK3 does not block CK1ε binding to Tcf3, whereas excess cold MBP-CKIε fails to block GSK3 binding to Tcf3, which suggests the existence of independent nonoverlapping sites for GSK3 and CK1ε binding on Tcf3. (D) CK1ε can phosphorylate Tcf3. Incubating Tcf3 with MBP-CK1ε enhances its phosphorylation. Endogenous kinase activity seen for Tcf3 alone reflects copurification of GSK3. (E) The enhancement of Tcf3 phosphorylation by CK1ε can be readily reversed by addition of CKI-7 (100 μM), a specific CK1 inhibitor, which indicates that Tcf3 is a substrate for both GSK3 and CK1ε. (F) Both GSK3 and CK1ε coimmunoprecipitates with myc-tagged Tcf3. Both cells of 2-cell embryos were injected with myc6-Tcf3 RNA (500 pg/blastomere), homogenized at stage 7.5, and precipitated with either anti-myc antibodies coupled to beads or to control beads. Western blotting with antibodies against GSK3 and CK1e indicates that both proteins coimmunoprecipitates with myc-tagged Tcf3 protein.
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fig6: GSK3 and CK1ε bind and phosphorylate Tcf3. (A) Tcf3 purified from Sf9 cells contains lithium-sensitive kinase activity. Recombinant his6-Tcf3 (1μg) was incubated in 10 μl kinase buffer (described in Materials and methods) for 30 min at room temperature either in the presence or absence of 100 mM LiCl, which normally inhibits GSK3 activity. Phosphorylation of Tcf3 is dramatically decreased in the presence of lithium. (B) Tcf3 beads pull down GSK3 from Xenopus extracts. Beads (control-BSA or Tcf3-coupled) were incubated with Xenopus egg extracts, washed, eluted, and analyzed by Western blotting with a monoclonal anti-GSK3 antibody. (C) Both GSK3 and CK1ε bind Tcf3. Radiolabeled in vitro–translated GSK3 and CK1ε bind Tcf3 beads. Binding of radiolabeled GSK3 and CK1ε to Tcf3 beads was abolished by addition of excess cold protein (5 μM of his6-GSK3 and MBP-CK1ε, respectively), demonstrating specificity of the binding reaction. However, excess cold his6-GSK3 does not block CK1ε binding to Tcf3, whereas excess cold MBP-CKIε fails to block GSK3 binding to Tcf3, which suggests the existence of independent nonoverlapping sites for GSK3 and CK1ε binding on Tcf3. (D) CK1ε can phosphorylate Tcf3. Incubating Tcf3 with MBP-CK1ε enhances its phosphorylation. Endogenous kinase activity seen for Tcf3 alone reflects copurification of GSK3. (E) The enhancement of Tcf3 phosphorylation by CK1ε can be readily reversed by addition of CKI-7 (100 μM), a specific CK1 inhibitor, which indicates that Tcf3 is a substrate for both GSK3 and CK1ε. (F) Both GSK3 and CK1ε coimmunoprecipitates with myc-tagged Tcf3. Both cells of 2-cell embryos were injected with myc6-Tcf3 RNA (500 pg/blastomere), homogenized at stage 7.5, and precipitated with either anti-myc antibodies coupled to beads or to control beads. Western blotting with antibodies against GSK3 and CK1e indicates that both proteins coimmunoprecipitates with myc-tagged Tcf3 protein.

Mentions: We noted during the characterization of recombinant histidine-tagged Tcf3 (his6-Tcf3) purified from baculovirus-infected Sf9 cells that certain preparations exhibited kinase activity, resulting in a phosphorylated form of Tcf3 that comigrated with the unphosphorylated form by SDS-PAGE. Furthermore, addition of recombinant his6-GSK3 to the reaction enhanced the phosphorylation of Tcf3 (unpublished data). Phosphorylation of Tcf3 was suppressed by 100 mM LiCl (a direct inhibitor of GSK3) (Klein and Melton, 1996) to the reaction (Fig. 6 A), which suggested that endogenous GSK3 copurified with recombinant his6-Tcf3 from Sf9 cells. This was confirmed by Western analysis using an anti-GSK3 antibody (unpublished data). To rule out the possibility that GSK3 gratuitously copurified with Tcf3 on the Ni2+ beads (which have ion exchange properties), we tested whether Tcf3 beads could be used to purify GSK3 from Xenopus extracts. We found that Tcf3 beads but not control BSA beads were capable of purifying GSK3 from Xenopus extracts (Fig. 6 B). No GSK3 was eluted from Tcf3 beads that were not preincubated in Xenopus extracts, which ruled out the possibility that the eluted GSK3 originated from Sf9 cells during the purification of Tcf3.


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

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

GSK3 and CK1ε bind and phosphorylate Tcf3. (A) Tcf3 purified from Sf9 cells contains lithium-sensitive kinase activity. Recombinant his6-Tcf3 (1μg) was incubated in 10 μl kinase buffer (described in Materials and methods) for 30 min at room temperature either in the presence or absence of 100 mM LiCl, which normally inhibits GSK3 activity. Phosphorylation of Tcf3 is dramatically decreased in the presence of lithium. (B) Tcf3 beads pull down GSK3 from Xenopus extracts. Beads (control-BSA or Tcf3-coupled) were incubated with Xenopus egg extracts, washed, eluted, and analyzed by Western blotting with a monoclonal anti-GSK3 antibody. (C) Both GSK3 and CK1ε bind Tcf3. Radiolabeled in vitro–translated GSK3 and CK1ε bind Tcf3 beads. Binding of radiolabeled GSK3 and CK1ε to Tcf3 beads was abolished by addition of excess cold protein (5 μM of his6-GSK3 and MBP-CK1ε, respectively), demonstrating specificity of the binding reaction. However, excess cold his6-GSK3 does not block CK1ε binding to Tcf3, whereas excess cold MBP-CKIε fails to block GSK3 binding to Tcf3, which suggests the existence of independent nonoverlapping sites for GSK3 and CK1ε binding on Tcf3. (D) CK1ε can phosphorylate Tcf3. Incubating Tcf3 with MBP-CK1ε enhances its phosphorylation. Endogenous kinase activity seen for Tcf3 alone reflects copurification of GSK3. (E) The enhancement of Tcf3 phosphorylation by CK1ε can be readily reversed by addition of CKI-7 (100 μM), a specific CK1 inhibitor, which indicates that Tcf3 is a substrate for both GSK3 and CK1ε. (F) Both GSK3 and CK1ε coimmunoprecipitates with myc-tagged Tcf3. Both cells of 2-cell embryos were injected with myc6-Tcf3 RNA (500 pg/blastomere), homogenized at stage 7.5, and precipitated with either anti-myc antibodies coupled to beads or to control beads. Western blotting with antibodies against GSK3 and CK1e indicates that both proteins coimmunoprecipitates with myc-tagged Tcf3 protein.
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Related In: Results  -  Collection

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fig6: GSK3 and CK1ε bind and phosphorylate Tcf3. (A) Tcf3 purified from Sf9 cells contains lithium-sensitive kinase activity. Recombinant his6-Tcf3 (1μg) was incubated in 10 μl kinase buffer (described in Materials and methods) for 30 min at room temperature either in the presence or absence of 100 mM LiCl, which normally inhibits GSK3 activity. Phosphorylation of Tcf3 is dramatically decreased in the presence of lithium. (B) Tcf3 beads pull down GSK3 from Xenopus extracts. Beads (control-BSA or Tcf3-coupled) were incubated with Xenopus egg extracts, washed, eluted, and analyzed by Western blotting with a monoclonal anti-GSK3 antibody. (C) Both GSK3 and CK1ε bind Tcf3. Radiolabeled in vitro–translated GSK3 and CK1ε bind Tcf3 beads. Binding of radiolabeled GSK3 and CK1ε to Tcf3 beads was abolished by addition of excess cold protein (5 μM of his6-GSK3 and MBP-CK1ε, respectively), demonstrating specificity of the binding reaction. However, excess cold his6-GSK3 does not block CK1ε binding to Tcf3, whereas excess cold MBP-CKIε fails to block GSK3 binding to Tcf3, which suggests the existence of independent nonoverlapping sites for GSK3 and CK1ε binding on Tcf3. (D) CK1ε can phosphorylate Tcf3. Incubating Tcf3 with MBP-CK1ε enhances its phosphorylation. Endogenous kinase activity seen for Tcf3 alone reflects copurification of GSK3. (E) The enhancement of Tcf3 phosphorylation by CK1ε can be readily reversed by addition of CKI-7 (100 μM), a specific CK1 inhibitor, which indicates that Tcf3 is a substrate for both GSK3 and CK1ε. (F) Both GSK3 and CK1ε coimmunoprecipitates with myc-tagged Tcf3. Both cells of 2-cell embryos were injected with myc6-Tcf3 RNA (500 pg/blastomere), homogenized at stage 7.5, and precipitated with either anti-myc antibodies coupled to beads or to control beads. Western blotting with antibodies against GSK3 and CK1e indicates that both proteins coimmunoprecipitates with myc-tagged Tcf3 protein.
Mentions: We noted during the characterization of recombinant histidine-tagged Tcf3 (his6-Tcf3) purified from baculovirus-infected Sf9 cells that certain preparations exhibited kinase activity, resulting in a phosphorylated form of Tcf3 that comigrated with the unphosphorylated form by SDS-PAGE. Furthermore, addition of recombinant his6-GSK3 to the reaction enhanced the phosphorylation of Tcf3 (unpublished data). Phosphorylation of Tcf3 was suppressed by 100 mM LiCl (a direct inhibitor of GSK3) (Klein and Melton, 1996) to the reaction (Fig. 6 A), which suggested that endogenous GSK3 copurified with recombinant his6-Tcf3 from Sf9 cells. This was confirmed by Western analysis using an anti-GSK3 antibody (unpublished data). To rule out the possibility that GSK3 gratuitously copurified with Tcf3 on the Ni2+ beads (which have ion exchange properties), we tested whether Tcf3 beads could be used to purify GSK3 from Xenopus extracts. We found that Tcf3 beads but not control BSA beads were capable of purifying GSK3 from Xenopus extracts (Fig. 6 B). No GSK3 was eluted from Tcf3 beads that were not preincubated in Xenopus extracts, which ruled out the possibility that the eluted GSK3 originated from Sf9 cells during the purification of Tcf3.

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