Limits...
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.

Show MeSH

Related in: MedlinePlus

Effect of CK1ε on Tcf3– β-catenin interaction. (A) Tcf3 and CK1ε act synergistically to inhibit β-catenin degradation. His6-Tcf3 (3 nM) and MBP-CK1ε (200 nM) were added to Xenopus extracts either alone or together. Inhibition of β-catenin degradation is dramatically enhanced by addition of both Tcf3 and CK1ε (nearly 80% remaining after 3 h) compared with addition of either Tcf3 or CK1ε alone (35% remaining after 3 h). (B) The CK1ε inhibitor CKI-7 inhibits the effect of Tcf3 or β-catenin stabilization. CKI-7 inhibits the effects of 10 and 30 nM Tcf3 in a dose-dependent manner. At high CKI-7 concentrations (100 μM), the effect of Tcf3 is abolished and β-catenin degradation is actually stimulated when compared with the buffer control. (C) CK1ε stimulates the binding of Tcf3 to β-catenin. Preincubation of Tcf3 beads with 1 μM CK1ε in kinase buffer stimulates its binding to β-catenin (compared with preincubation in buffer alone). This effect of CK1ε was decreased by addition of 1 μM GSK3 to the kinase reaction. GSK3 by itself had no effect on the binding of Tcf3 to β-catenin. A nearly fivefold increase in the binding of Tcf3 to β-catenin is seen when Tcf3 beads were preincubated with CK1ε. (D) CK1ε acts synergistically with GBP to inhibit β-catenin degradation. 50 nM GBP or 200 nM CK1ε has no effect on β-catenin degradation; however, together they dramatically inhibit β-catenin degradation in extracts.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2196183&req=5

fig7: Effect of CK1ε on Tcf3– β-catenin interaction. (A) Tcf3 and CK1ε act synergistically to inhibit β-catenin degradation. His6-Tcf3 (3 nM) and MBP-CK1ε (200 nM) were added to Xenopus extracts either alone or together. Inhibition of β-catenin degradation is dramatically enhanced by addition of both Tcf3 and CK1ε (nearly 80% remaining after 3 h) compared with addition of either Tcf3 or CK1ε alone (35% remaining after 3 h). (B) The CK1ε inhibitor CKI-7 inhibits the effect of Tcf3 or β-catenin stabilization. CKI-7 inhibits the effects of 10 and 30 nM Tcf3 in a dose-dependent manner. At high CKI-7 concentrations (100 μM), the effect of Tcf3 is abolished and β-catenin degradation is actually stimulated when compared with the buffer control. (C) CK1ε stimulates the binding of Tcf3 to β-catenin. Preincubation of Tcf3 beads with 1 μM CK1ε in kinase buffer stimulates its binding to β-catenin (compared with preincubation in buffer alone). This effect of CK1ε was decreased by addition of 1 μM GSK3 to the kinase reaction. GSK3 by itself had no effect on the binding of Tcf3 to β-catenin. A nearly fivefold increase in the binding of Tcf3 to β-catenin is seen when Tcf3 beads were preincubated with CK1ε. (D) CK1ε acts synergistically with GBP to inhibit β-catenin degradation. 50 nM GBP or 200 nM CK1ε has no effect on β-catenin degradation; however, together they dramatically inhibit β-catenin degradation in extracts.

Mentions: These experiments establish that both GSK3 and CK1ε can bind and phosphorylate Tcf3 and suggest a possible role for both GSK3 and CK1ε in modulating Tcf3 activity. We therefore tested if these kinases affect the interaction of Tcf3 with β-catenin. Tcf3 can inhibit the degradation of β-catenin (Fig. 1, A and B). CK1ε and Tcf3 act synergistically to inhibit β-catenin degradation: as shown in Fig. 7 A, concentrations of CK1ε and Tcf3 that by themselves are not inhibitory together cause significant inhibition of β-catenin degradation. In addition, CKI-7 blocks the effect of Tcf3 in a dose-dependent manner in Xenopus extracts (Fig. 7 B). As expected, 100 μM CKI-7 (even in the presence of 10 nM Tcf3) actually accelerates the rate of degradation of β-catenin in extracts compared with a buffer control. Thus, CK1ε activity is required for inhibition of β-catenin degradation by Tcf3. The simplest model consistent with these experimental results is that phosphorylation of Tcf3 by CK1ε promotes the interaction of Tcf3 with β-catenin. As shown in Fig. 7 C, preincubation of Tcf3 beads with CK1ε increases their affinity for β-catenin nearly fourfold compared with untreated beads. Furthermore, although preincubating Tcf3 beads with GSK3 had very little effect on β-catenin binding, GSK3 abolished the effect of CK1ε. One prediction from these experiments is that blocking GSK3 activity would further potentiate the effect of CK1ε. As expected, GBP acts synergistically with CK1ε to inhibit β-catenin degradation (Fig. 7 D). Failure of GSK3 to inhibit the effects of Tcf3 in our earlier experiments (Fig. 1 B) was probably due to the high concentration of Tcf3 (1 μM) used. In fact, the effect of adding Tcf3 at concentrations closer to its IC50 for β-catenin degradation (100 nM) can be reversed by the addition of 1 μM GSK3 (Fig. 1 C).


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

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

Effect of CK1ε on Tcf3– β-catenin interaction. (A) Tcf3 and CK1ε act synergistically to inhibit β-catenin degradation. His6-Tcf3 (3 nM) and MBP-CK1ε (200 nM) were added to Xenopus extracts either alone or together. Inhibition of β-catenin degradation is dramatically enhanced by addition of both Tcf3 and CK1ε (nearly 80% remaining after 3 h) compared with addition of either Tcf3 or CK1ε alone (35% remaining after 3 h). (B) The CK1ε inhibitor CKI-7 inhibits the effect of Tcf3 or β-catenin stabilization. CKI-7 inhibits the effects of 10 and 30 nM Tcf3 in a dose-dependent manner. At high CKI-7 concentrations (100 μM), the effect of Tcf3 is abolished and β-catenin degradation is actually stimulated when compared with the buffer control. (C) CK1ε stimulates the binding of Tcf3 to β-catenin. Preincubation of Tcf3 beads with 1 μM CK1ε in kinase buffer stimulates its binding to β-catenin (compared with preincubation in buffer alone). This effect of CK1ε was decreased by addition of 1 μM GSK3 to the kinase reaction. GSK3 by itself had no effect on the binding of Tcf3 to β-catenin. A nearly fivefold increase in the binding of Tcf3 to β-catenin is seen when Tcf3 beads were preincubated with CK1ε. (D) CK1ε acts synergistically with GBP to inhibit β-catenin degradation. 50 nM GBP or 200 nM CK1ε has no effect on β-catenin degradation; however, together they dramatically inhibit β-catenin degradation in extracts.
© Copyright Policy
Related In: Results  -  Collection

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

fig7: Effect of CK1ε on Tcf3– β-catenin interaction. (A) Tcf3 and CK1ε act synergistically to inhibit β-catenin degradation. His6-Tcf3 (3 nM) and MBP-CK1ε (200 nM) were added to Xenopus extracts either alone or together. Inhibition of β-catenin degradation is dramatically enhanced by addition of both Tcf3 and CK1ε (nearly 80% remaining after 3 h) compared with addition of either Tcf3 or CK1ε alone (35% remaining after 3 h). (B) The CK1ε inhibitor CKI-7 inhibits the effect of Tcf3 or β-catenin stabilization. CKI-7 inhibits the effects of 10 and 30 nM Tcf3 in a dose-dependent manner. At high CKI-7 concentrations (100 μM), the effect of Tcf3 is abolished and β-catenin degradation is actually stimulated when compared with the buffer control. (C) CK1ε stimulates the binding of Tcf3 to β-catenin. Preincubation of Tcf3 beads with 1 μM CK1ε in kinase buffer stimulates its binding to β-catenin (compared with preincubation in buffer alone). This effect of CK1ε was decreased by addition of 1 μM GSK3 to the kinase reaction. GSK3 by itself had no effect on the binding of Tcf3 to β-catenin. A nearly fivefold increase in the binding of Tcf3 to β-catenin is seen when Tcf3 beads were preincubated with CK1ε. (D) CK1ε acts synergistically with GBP to inhibit β-catenin degradation. 50 nM GBP or 200 nM CK1ε has no effect on β-catenin degradation; however, together they dramatically inhibit β-catenin degradation in extracts.
Mentions: These experiments establish that both GSK3 and CK1ε can bind and phosphorylate Tcf3 and suggest a possible role for both GSK3 and CK1ε in modulating Tcf3 activity. We therefore tested if these kinases affect the interaction of Tcf3 with β-catenin. Tcf3 can inhibit the degradation of β-catenin (Fig. 1, A and B). CK1ε and Tcf3 act synergistically to inhibit β-catenin degradation: as shown in Fig. 7 A, concentrations of CK1ε and Tcf3 that by themselves are not inhibitory together cause significant inhibition of β-catenin degradation. In addition, CKI-7 blocks the effect of Tcf3 in a dose-dependent manner in Xenopus extracts (Fig. 7 B). As expected, 100 μM CKI-7 (even in the presence of 10 nM Tcf3) actually accelerates the rate of degradation of β-catenin in extracts compared with a buffer control. Thus, CK1ε activity is required for inhibition of β-catenin degradation by Tcf3. The simplest model consistent with these experimental results is that phosphorylation of Tcf3 by CK1ε promotes the interaction of Tcf3 with β-catenin. As shown in Fig. 7 C, preincubation of Tcf3 beads with CK1ε increases their affinity for β-catenin nearly fourfold compared with untreated beads. Furthermore, although preincubating Tcf3 beads with GSK3 had very little effect on β-catenin binding, GSK3 abolished the effect of CK1ε. One prediction from these experiments is that blocking GSK3 activity would further potentiate the effect of CK1ε. As expected, GBP acts synergistically with CK1ε to inhibit β-catenin degradation (Fig. 7 D). Failure of GSK3 to inhibit the effects of Tcf3 in our earlier experiments (Fig. 1 B) was probably due to the high concentration of Tcf3 (1 μM) used. In fact, the effect of adding Tcf3 at concentrations closer to its IC50 for β-catenin degradation (100 nM) can be reversed by the addition of 1 μM GSK3 (Fig. 1 C).

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