Lee E et al. (2001), Physiological regulation of [beta]-catenin stab...

XB-ART-8496

J Cell Biol 2001 Sep 03;1545:983-93. doi: 10.1083/jcb.200102074.

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

Lee E , Salic A , Kirschner MW .

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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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Species referenced: Xenopus laevis
Genes referenced: csnk1a1 csnk1e csnk1g2 ctnnb1 dvl1 dvl2 frat1 gsk3b herpud1 krt8.1 mbp myc tcf3 tcf4 tcf7l1

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	Figure 1. Tcf3 blocks β-catenin degradation in extracts and phosphorylation in vitro. (A) Translated Tcf3 but not ΔNTcf3 mRNA inhibits β-catenin degradation in extracts. (B) Purified Tcf3 protein (1 μM) blocks β-catenin degradation. This effect was not reversed by 1 μM GSK3. Purified ΔNTcf3 (1 μM) does not block β-catenin degradation. (C) Inhibition of β-catenin degradation by lower (100 nM) Tcf3 levels can be partially rescued by GSK3; higher Tcf3 (300 nM) levels cannot be rescued by even a large GSK3 excess. (D) Tcf3 inhibits the phosphorylation of β-catenin by GSK3 and axin in a purified system. In the same reaction, axin phosphorylation by GSK3 is not affected by Tcf3.
	Figure 2. 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).
	Figure 3. 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.
	Figure 4. Effects of an NH2-terminal Tcf4 peptide on β-catenin stability and rate of axin–β-catenin dissociation. (A) β-catenin binding to xTcf3 beads is inhibited by an NH2-terminal Tcf4 peptide (10 μM). (B) In contrast, β-catenin binding to axin is unaffected by the presence of the NH2-terminal Tcf4 peptide but is blocked by cold Tcf3. (C) Addition of the NH2-terminal Tcf4 peptide (2 μM) to Xenopus extracts stimulates β-catenin degradation, which is in contrast to the inhibitory effect of Tcf3 (500 nM). (D) Half-life of the axin–β-catenin complex. Radiolabeled β-catenin bound to axin beads (in the presence or absence of 100 nM APC) was incubated with buffer, cold Tcf3 (2 μM) or cold β-catenin (2 μM) at room temperature. The β-catenin remaining on beads was measured as a function of time.
	Figure 5. 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.
	Figure 6. 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.
	Figure 7. 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.
	Figure 8. Role of CK1ε in mediating dsh activity. (A) dsh acts synergistically with CK1ε in Xenopus embryos to induce axis duplication. Embryos were injected with 50 pg CK1ε, 100 pg dsh, or 50 pg CK1ε plus 100 pg dsh RNA in one ventral blastomere of 4–8-cell stage embryos. (B) Dsh and CK1e act synergistically to inhibit β-catenin degradation. Dsh protein (100 nM) and CK1e protein (500 nM) were added to extracts alone or in combination. (C) GBP cross-linked beads were incubated with radiolabeled dsh and either buffer, 1 μM CK1ε, or 1 μM GSK3 in the presence or absence of Xenopus extracts. CK1ε stimulates the binding of GBP to radiolabeled dsh in Xenopus extracts. No differences were detected when binding was performed in the absence of extracts.

References [+] :

Aberle, beta-catenin is a target for the ubiquitin-proteasome pathway. 1997, Pubmed