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.

Show Gene links Show Anatomy links

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

Salic A , Kirschner MW .

???displayArticle.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.

???displayArticle.pubmedLink??? 11524435
???displayArticle.pmcLink??? PMC2196183
???displayArticle.link??? J Cell Biol

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

???attribute.lit??? ???displayArticles.show???

	Figure 1. Tcf3 blocks Î²-catenin degradation in extracts and phosphorylation in vitro. (A) Translated Tcf3 but not Î”NTcf3 mRNA inhibits Î²-catenin degradation in extracts. (B) Purified Tcf3 protein (1 Î¼M) blocks Î²-catenin degradation. This effect was not reversed by 1 Î¼M GSK3. Purified Î”NTcf3 (1 Î¼M) does not block Î²-catenin degradation. (C) Inhibition of Î²-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 Î²-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 Î²-catenin (cold competitors were present at 1 Î¼M in all experiments). (A) Binding of Î²-catenin to axin beads is inhibited by Tcf3 but not by Î”NTcf3. In contrast, binding of the Î²-catenin Î”C2 mutant to axin is unaffected by Tcf3. (B) Tcf3 blocks the binding of Î²-catenin to axin beads, whereas APCm3 reverses this effect of Tcf3. (C) APCm3 inhibits the binding of Î²-catenin to Tcf3 beads. (D) 1 Î¼M his6-TCF3 blocks the interaction of Î²-catenin with endogenous APC in extracts, whereas his6-Î”NTcf3 has no effect. (E) Scheme of the COOH-terminal Î²-catenin deletion constructs used to map the fragment of Î²-catenin responsible for stabilization by Tcf3. (F) Normal and axin-induced degradation of Î²-catenin, Î²-cateninÎ”C2, and Î²-cateninÎ”C3 in Xenopus extracts. Î²-cateninÎ”C3 is completely stable and does not respond to axin. (G) Axin stimulates the turnover of both Î²-catenin and Î²-cateninÎ”C2 in the same degradation reaction. (H) Î²-catenin and Î²-cateninÎ”C2 both bind to APC and binding is not disrupted by the Tcf4 NH2-terminal peptide or by the cat449/645 fragment (at <2 Î¼M).
	Figure 3. Effects of Tcf3 on Î²-catenin mutants. (A) Degradation of both Î²-catenin and Î²-cateninÎ”C2 is inhibited by 1 Î¼M dsh, but only Î²-catenin is inhibited by 1 Î¼M Tcf3. (B) Graphical representation of densitometry measurements of the autoradiogram in A shows the faster degradation rate of Î²-cateninÎ”C2 compared with Î²-catenin. (C) Both Î²-catenin and Î²-cateninÎ”C2 bind to axin, but only Î²-catenin binds to xTcf3 in vitro. (D) MBP-cat449/645 inhibits by >95% the binding of [35S]methionine-labeled Î²-catenin to Tcf3 in vitro (ii). MBP-cat449/645 (2 Î¼M) had no effect on the binding of axin to Î²-catenin beads (i) and a moderate effect on the binding of Î²-catenin to APC beads (iii). Binding of radiolabeled Î²-catenin and axin to control beads was negligible. (E) Degradation of radiolabeled Î²-catenin in Xenopus extracts is stimulated by MBP-cat449/645 (200 nM). (F) Î²-cateninâ€“luciferase is degraded more rapidly in embryos when coinjected with MBP-cat449/645. Î²-cateninâ€“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 Î²-catenin stability and rate of axinâ€“Î²-catenin dissociation. (A) Î²-catenin binding to xTcf3 beads is inhibited by an NH2-terminal Tcf4 peptide (10 Î¼M). (B) In contrast, Î²-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 Î¼M) to Xenopus extracts stimulates Î²-catenin degradation, which is in contrast to the inhibitory effect of Tcf3 (500 nM). (D) Half-life of the axinâ€“Î²-catenin complex. Radiolabeled Î²-catenin bound to axin beads (in the presence or absence of 100 nM APC) was incubated with buffer, cold Tcf3 (2 Î¼M) or cold Î²-catenin (2 Î¼M) at room temperature. The Î²-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 CK1Îµ bind and phosphorylate Tcf3. (A) Tcf3 purified from Sf9 cells contains lithium-sensitive kinase activity. Recombinant his6-Tcf3 (1Î¼g) was incubated in 10 Î¼l 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 CK1Îµ bind Tcf3. Radiolabeled in vitroâtranslated GSK3 and CK1Îµ bind Tcf3 beads. Binding of radiolabeled GSK3 and CK1Îµ to Tcf3 beads was abolished by addition of excess cold protein (5 Î¼M of his6-GSK3 and MBP-CK1Îµ, respectively), demonstrating specificity of the binding reaction. However, excess cold his6-GSK3 does not block CK1Îµ binding to Tcf3, whereas excess cold MBP-CKIÎµ fails to block GSK3 binding to Tcf3, which suggests the existence of independent nonoverlapping sites for GSK3 and CK1Îµ binding on Tcf3. (D) CK1Îµ can phosphorylate Tcf3. Incubating Tcf3 with MBP-CK1Îµ enhances its phosphorylation. Endogenous kinase activity seen for Tcf3 alone reflects copurification of GSK3. (E) The enhancement of Tcf3 phosphorylation by CK1Îµ can be readily reversed by addition of CKI-7 (100 Î¼M), a specific CK1 inhibitor, which indicates that Tcf3 is a substrate for both GSK3 and CK1Îµ. (F) Both GSK3 and CK1Îµ 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 CK1Îµ on Tcf3â€“ Î²-catenin interaction. (A) Tcf3 and CK1Îµ act synergistically to inhibit Î²-catenin degradation. His6-Tcf3 (3 nM) and MBP-CK1Îµ (200 nM) were added to Xenopus extracts either alone or together. Inhibition of Î²-catenin degradation is dramatically enhanced by addition of both Tcf3 and CK1Îµ (nearly 80% remaining after 3 h) compared with addition of either Tcf3 or CK1Îµ alone (35% remaining after 3 h). (B) The CK1Îµ inhibitor CKI-7 inhibits the effect of Tcf3 or Î²-catenin stabilization. CKI-7 inhibits the effects of 10 and 30 nM Tcf3 in a dose-dependent manner. At high CKI-7 concentrations (100 Î¼M), the effect of Tcf3 is abolished and Î²-catenin degradation is actually stimulated when compared with the buffer control. (C) CK1Îµ stimulates the binding of Tcf3 to Î²-catenin. Preincubation of Tcf3 beads with 1 Î¼M CK1Îµ in kinase buffer stimulates its binding to Î²-catenin (compared with preincubation in buffer alone). This effect of CK1Îµ was decreased by addition of 1 Î¼M GSK3 to the kinase reaction. GSK3 by itself had no effect on the binding of Tcf3 to Î²-catenin. A nearly fivefold increase in the binding of Tcf3 to Î²-catenin is seen when Tcf3 beads were preincubated with CK1Îµ. (D) CK1Îµ acts synergistically with GBP to inhibit Î²-catenin degradation. 50 nM GBP or 200 nM CK1Îµ has no effect on Î²-catenin degradation; however, together they dramatically inhibit Î²-catenin degradation in extracts.
	Figure 8. Role of CK1Îµ in mediating dsh activity. (A) dsh acts synergistically with CK1Îµ in Xenopus embryos to induce axis duplication. Embryos were injected with 50 pg CK1Îµ, 100 pg dsh, or 50 pg CK1Îµ plus 100 pg dsh RNA in one ventral blastomere of 4â€“8-cell stage embryos. (B) Dsh and CK1e act synergistically to inhibit Î²-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 Î¼M CK1Îµ, or 1 Î¼M GSK3 in the presence or absence of Xenopus extracts. CK1Îµ 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