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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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A significant fraction of total cellular TCF protein in Xenopus embryos and in cultured cells is nonnuclear. (A) Both cells of 2-cell embryos were injected with myc6-Tcf3 RNA (250 pg/ blastomere), gently homogenized at stage 7.5 (lysate), and centrifuged to pellet the nuclei (Sup). Equivalent volumes of lysate and supernatant were subsequently processed for Western analysis using an anti-myc antibody. Most of the detected myc-tagged Tcf3 is present in the supernatant fraction. (B) Xenopus eggs and stage 7.5 embryos were processed as described for the myc6-Tcf3 RNA injected embryos except that an anti-Tcf antibody was used to detect endogenous Tcf. Nearly all of the Tcf detected is present in the supernatant fraction of eggs in contrast to stage 7.5 embryos. Lysates and supernatants were stained with Hoechst to confirm the presence (lysates) or absence (supernatants) of intact nuclei (unpublished data). Nuclear and cytoplasmic preparations from cultured 293 cells were blotted for topoisomerase II (C) and Tcf and stained with Hoechst (D). The nuclear pellet was brought to the same volume as the cytoplasmic fraction, and equivalent volumes were used for Western analysis.
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fig5: A significant fraction of total cellular TCF protein in Xenopus embryos and in cultured cells is nonnuclear. (A) Both cells of 2-cell embryos were injected with myc6-Tcf3 RNA (250 pg/ blastomere), gently homogenized at stage 7.5 (lysate), and centrifuged to pellet the nuclei (Sup). Equivalent volumes of lysate and supernatant were subsequently processed for Western analysis using an anti-myc antibody. Most of the detected myc-tagged Tcf3 is present in the supernatant fraction. (B) Xenopus eggs and stage 7.5 embryos were processed as described for the myc6-Tcf3 RNA injected embryos except that an anti-Tcf antibody was used to detect endogenous Tcf. Nearly all of the Tcf detected is present in the supernatant fraction of eggs in contrast to stage 7.5 embryos. Lysates and supernatants were stained with Hoechst to confirm the presence (lysates) or absence (supernatants) of intact nuclei (unpublished data). Nuclear and cytoplasmic preparations from cultured 293 cells were blotted for topoisomerase II (C) and Tcf and stained with Hoechst (D). The nuclear pellet was brought to the same volume as the cytoplasmic fraction, and equivalent volumes were used for Western analysis.

Mentions: Although we have demonstrated that Tcf proteins can regulate the rate of β-catenin degradation, if Tcf and the proteins involved in β-catenin turnover do not normally colocalize in the cell such a mechanism might not be physiological. Specifically, Tcf is a transcription factor that may reside exclusively in the nucleus, whereas β-catenin degradation may occur solely in the cytoplasm. Therefore, we measured the amount of cytoplasmic and nuclear Tcf in Xenopus eggs, stage 7.5 Xenopus embryos, and cultured human 293 cells. As shown in Fig. 5 B, the vast majority of Tcf is cytoplasmic in eggs. Even in stage 7.5 embryos where the number of nuclei is ∼2,000 per embryo (versus one per egg), a large fraction of Tcf is cytoplasmic (Fig. 5 A; 70% cytoplasmic fraction versus 30% in the nuclear fraction). We found a similar distribution of myc6-tagged xTcf3 protein in stage 7.5 embryos that were injected with myc6-tagged xTcf3 mRNA at the 2-cell stage. These findings are perhaps not surprising and may reflect two conditions found in a developing embryo: (a) the low nucleo-cytoplasmic ratio of the embryonic cells and (2) the rapid division cycles of the early embryo (30 min per cycle), which results in disassembly of the nucleus for half of the time. These two situations allow ample opportunity for Tcf to interact with cytoplasmic β-catenin to regulate its degradation. We next wanted to determine whether or not the high level of cytoplasmic Tcf protein was unique to Xenopus embryos and examined the cytoplasmic pool of Tcf in cultured cells. As shown in Fig. 5, C and D, fractionation of 293 cells indicates that ∼40% of Tcf was cytoplasmic (compared with topoisomerase II, which is quantitatively pelleted with the nuclei). Therefore, in cultured 293 cells a significant amount of Tcf is present in the cytoplasm and thus capable of competing with APC and axin for β-catenin binding.


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

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

A significant fraction of total cellular TCF protein in Xenopus embryos and in cultured cells is nonnuclear. (A) Both cells of 2-cell embryos were injected with myc6-Tcf3 RNA (250 pg/ blastomere), gently homogenized at stage 7.5 (lysate), and centrifuged to pellet the nuclei (Sup). Equivalent volumes of lysate and supernatant were subsequently processed for Western analysis using an anti-myc antibody. Most of the detected myc-tagged Tcf3 is present in the supernatant fraction. (B) Xenopus eggs and stage 7.5 embryos were processed as described for the myc6-Tcf3 RNA injected embryos except that an anti-Tcf antibody was used to detect endogenous Tcf. Nearly all of the Tcf detected is present in the supernatant fraction of eggs in contrast to stage 7.5 embryos. Lysates and supernatants were stained with Hoechst to confirm the presence (lysates) or absence (supernatants) of intact nuclei (unpublished data). Nuclear and cytoplasmic preparations from cultured 293 cells were blotted for topoisomerase II (C) and Tcf and stained with Hoechst (D). The nuclear pellet was brought to the same volume as the cytoplasmic fraction, and equivalent volumes were used for Western analysis.
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Related In: Results  -  Collection

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fig5: A significant fraction of total cellular TCF protein in Xenopus embryos and in cultured cells is nonnuclear. (A) Both cells of 2-cell embryos were injected with myc6-Tcf3 RNA (250 pg/ blastomere), gently homogenized at stage 7.5 (lysate), and centrifuged to pellet the nuclei (Sup). Equivalent volumes of lysate and supernatant were subsequently processed for Western analysis using an anti-myc antibody. Most of the detected myc-tagged Tcf3 is present in the supernatant fraction. (B) Xenopus eggs and stage 7.5 embryos were processed as described for the myc6-Tcf3 RNA injected embryos except that an anti-Tcf antibody was used to detect endogenous Tcf. Nearly all of the Tcf detected is present in the supernatant fraction of eggs in contrast to stage 7.5 embryos. Lysates and supernatants were stained with Hoechst to confirm the presence (lysates) or absence (supernatants) of intact nuclei (unpublished data). Nuclear and cytoplasmic preparations from cultured 293 cells were blotted for topoisomerase II (C) and Tcf and stained with Hoechst (D). The nuclear pellet was brought to the same volume as the cytoplasmic fraction, and equivalent volumes were used for Western analysis.
Mentions: Although we have demonstrated that Tcf proteins can regulate the rate of β-catenin degradation, if Tcf and the proteins involved in β-catenin turnover do not normally colocalize in the cell such a mechanism might not be physiological. Specifically, Tcf is a transcription factor that may reside exclusively in the nucleus, whereas β-catenin degradation may occur solely in the cytoplasm. Therefore, we measured the amount of cytoplasmic and nuclear Tcf in Xenopus eggs, stage 7.5 Xenopus embryos, and cultured human 293 cells. As shown in Fig. 5 B, the vast majority of Tcf is cytoplasmic in eggs. Even in stage 7.5 embryos where the number of nuclei is ∼2,000 per embryo (versus one per egg), a large fraction of Tcf is cytoplasmic (Fig. 5 A; 70% cytoplasmic fraction versus 30% in the nuclear fraction). We found a similar distribution of myc6-tagged xTcf3 protein in stage 7.5 embryos that were injected with myc6-tagged xTcf3 mRNA at the 2-cell stage. These findings are perhaps not surprising and may reflect two conditions found in a developing embryo: (a) the low nucleo-cytoplasmic ratio of the embryonic cells and (2) the rapid division cycles of the early embryo (30 min per cycle), which results in disassembly of the nucleus for half of the time. These two situations allow ample opportunity for Tcf to interact with cytoplasmic β-catenin to regulate its degradation. We next wanted to determine whether or not the high level of cytoplasmic Tcf protein was unique to Xenopus embryos and examined the cytoplasmic pool of Tcf in cultured cells. As shown in Fig. 5, C and D, fractionation of 293 cells indicates that ∼40% of Tcf was cytoplasmic (compared with topoisomerase II, which is quantitatively pelleted with the nuclei). Therefore, in cultured 293 cells a significant amount of Tcf is present in the cytoplasm and thus capable of competing with APC and axin for β-catenin binding.

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