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Wnt-5a inhibits the canonical Wnt pathway by promoting GSK-3-independent beta-catenin degradation.

Topol L, Jiang X, Choi H, Garrett-Beal L, Carolan PJ, Yang Y - J. Cell Biol. (2003)

Bottom Line: Wnt-5a is considered a noncanonical Wnt as it does not signal by stabilizing beta-catenin in many biological systems.We have uncovered a new noncanonical pathway through which Wnt-5a antagonizes the canonical Wnt pathway by promoting the degradation of beta-catenin.This pathway is Siah2 and APC dependent, but GSK-3 and beta-TrCP independent.

View Article: PubMed Central - PubMed

Affiliation: Genetic Disease Research Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892, USA.

ABSTRACT
Wnts are secreted signaling molecules that can transduce their signals through several different pathways. Wnt-5a is considered a noncanonical Wnt as it does not signal by stabilizing beta-catenin in many biological systems. We have uncovered a new noncanonical pathway through which Wnt-5a antagonizes the canonical Wnt pathway by promoting the degradation of beta-catenin. This pathway is Siah2 and APC dependent, but GSK-3 and beta-TrCP independent. Furthermore, we provide evidence that Wnt-5a also acts in vivo to promote beta-catenin degradation in regulating mammalian limb development and possibly in suppressing tumor formation.

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Wnt-5a promoted β-catenin degradation through a mechanism independent of GSK-3 and β-TrCP. (A) 24 h after transfection, 293 cells were treated with 40 mM LiCl for 16 h before cells were harvested. LiCl treatment up-regulated TOPFLASH reporter activity. Wnt-5a suppressed the effect of LiCl. (B) Cells were transfected with the indicated plasmids and harvested 48 h later for luciferase assay. Δβ-TrCP up-regulated the TOPFLASH reporter activity and this effect was suppressed by Wnt-5a. (C) 293 cells were transfected with the indicated plasmids and treated with LiCl as in A. LiCl treatment or Δβ-TrCP expression stabilized the endogenous β-catenin and Wnt-5a inhibited this effect. (D) Wnt-5a promoted the degradation of two mutant stabilized forms of β-catenin in 293 cells. Cells were transfected with the indicated plasmids and 48 h after transfection, cells were harvested for western analysis.
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fig2: Wnt-5a promoted β-catenin degradation through a mechanism independent of GSK-3 and β-TrCP. (A) 24 h after transfection, 293 cells were treated with 40 mM LiCl for 16 h before cells were harvested. LiCl treatment up-regulated TOPFLASH reporter activity. Wnt-5a suppressed the effect of LiCl. (B) Cells were transfected with the indicated plasmids and harvested 48 h later for luciferase assay. Δβ-TrCP up-regulated the TOPFLASH reporter activity and this effect was suppressed by Wnt-5a. (C) 293 cells were transfected with the indicated plasmids and treated with LiCl as in A. LiCl treatment or Δβ-TrCP expression stabilized the endogenous β-catenin and Wnt-5a inhibited this effect. (D) Wnt-5a promoted the degradation of two mutant stabilized forms of β-catenin in 293 cells. Cells were transfected with the indicated plasmids and 48 h after transfection, cells were harvested for western analysis.

Mentions: Because phosphorylation of β-catenin by GSK-3 followed by β-TrCP binding results in β-catenin degradation, we then tested whether Wnt-5a promoted β-catenin degradation requires GSK-3 or β-TrCP. We used LiCl to specifically inhibit GSK-3 (Klein and Melton, 1996). LiCl treatment resulted in the enhancement of TOPFLASH reporter activity and accumulation of endogenous β-catenin in 293 cells (Fig. 2, A and C). However, Wnt-5a was still able to inhibit β-catenin activity substantially in the presence of LiCl (Fig. 2 A). In addition, when the activity of β-TrCP was blocked by a dominant negative β-TrCP (Δβ-TrCP), Wnt-5a was still able to suppress β-catenin activity (Fig. 2 B). Consistent with the decrease in TOPFLASH activity, Wnt-5a signaling reduced endogenous β-catenin protein levels in the presence of LiCl and Δβ-TrCP (Fig. 2 C). These results indicate that the β-catenin protein degradation promoted by Wnt-5a was independent of GSK-3 and β-TrCP. Therefore, Wnt-5a might potentially be able to promote the degradation of two different forms of mutant β-catenin, which cannot be phosphorylated by GSK-3 and are constitutively active. One mutant, β-catenin S37A, is a Ser to Ala mutation that abolishes the GSK-3 dependent phosphorylation of β-catenin at Ser 37, which is required for β-TrCP binding. A second mutant, Δβ-catenin, lacks amino acids 29–48 and contains none of the GSK-3 phosphorylation sites that are required for β-catenin degradation (Tetsu and McCormick, 1999). Both mutants stabilized β-catenin. However, when they were coexpressed with Wnt-5a in 293 cells, we found that they were destabilized by Wnt-5a (Fig. 2 D), demonstrating that Wnt-5a functions to inhibit canonical Wnt/β-catenin pathway independently of GSK-3 and β-TrCP.


Wnt-5a inhibits the canonical Wnt pathway by promoting GSK-3-independent beta-catenin degradation.

Topol L, Jiang X, Choi H, Garrett-Beal L, Carolan PJ, Yang Y - J. Cell Biol. (2003)

Wnt-5a promoted β-catenin degradation through a mechanism independent of GSK-3 and β-TrCP. (A) 24 h after transfection, 293 cells were treated with 40 mM LiCl for 16 h before cells were harvested. LiCl treatment up-regulated TOPFLASH reporter activity. Wnt-5a suppressed the effect of LiCl. (B) Cells were transfected with the indicated plasmids and harvested 48 h later for luciferase assay. Δβ-TrCP up-regulated the TOPFLASH reporter activity and this effect was suppressed by Wnt-5a. (C) 293 cells were transfected with the indicated plasmids and treated with LiCl as in A. LiCl treatment or Δβ-TrCP expression stabilized the endogenous β-catenin and Wnt-5a inhibited this effect. (D) Wnt-5a promoted the degradation of two mutant stabilized forms of β-catenin in 293 cells. Cells were transfected with the indicated plasmids and 48 h after transfection, cells were harvested for western analysis.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172823&req=5

fig2: Wnt-5a promoted β-catenin degradation through a mechanism independent of GSK-3 and β-TrCP. (A) 24 h after transfection, 293 cells were treated with 40 mM LiCl for 16 h before cells were harvested. LiCl treatment up-regulated TOPFLASH reporter activity. Wnt-5a suppressed the effect of LiCl. (B) Cells were transfected with the indicated plasmids and harvested 48 h later for luciferase assay. Δβ-TrCP up-regulated the TOPFLASH reporter activity and this effect was suppressed by Wnt-5a. (C) 293 cells were transfected with the indicated plasmids and treated with LiCl as in A. LiCl treatment or Δβ-TrCP expression stabilized the endogenous β-catenin and Wnt-5a inhibited this effect. (D) Wnt-5a promoted the degradation of two mutant stabilized forms of β-catenin in 293 cells. Cells were transfected with the indicated plasmids and 48 h after transfection, cells were harvested for western analysis.
Mentions: Because phosphorylation of β-catenin by GSK-3 followed by β-TrCP binding results in β-catenin degradation, we then tested whether Wnt-5a promoted β-catenin degradation requires GSK-3 or β-TrCP. We used LiCl to specifically inhibit GSK-3 (Klein and Melton, 1996). LiCl treatment resulted in the enhancement of TOPFLASH reporter activity and accumulation of endogenous β-catenin in 293 cells (Fig. 2, A and C). However, Wnt-5a was still able to inhibit β-catenin activity substantially in the presence of LiCl (Fig. 2 A). In addition, when the activity of β-TrCP was blocked by a dominant negative β-TrCP (Δβ-TrCP), Wnt-5a was still able to suppress β-catenin activity (Fig. 2 B). Consistent with the decrease in TOPFLASH activity, Wnt-5a signaling reduced endogenous β-catenin protein levels in the presence of LiCl and Δβ-TrCP (Fig. 2 C). These results indicate that the β-catenin protein degradation promoted by Wnt-5a was independent of GSK-3 and β-TrCP. Therefore, Wnt-5a might potentially be able to promote the degradation of two different forms of mutant β-catenin, which cannot be phosphorylated by GSK-3 and are constitutively active. One mutant, β-catenin S37A, is a Ser to Ala mutation that abolishes the GSK-3 dependent phosphorylation of β-catenin at Ser 37, which is required for β-TrCP binding. A second mutant, Δβ-catenin, lacks amino acids 29–48 and contains none of the GSK-3 phosphorylation sites that are required for β-catenin degradation (Tetsu and McCormick, 1999). Both mutants stabilized β-catenin. However, when they were coexpressed with Wnt-5a in 293 cells, we found that they were destabilized by Wnt-5a (Fig. 2 D), demonstrating that Wnt-5a functions to inhibit canonical Wnt/β-catenin pathway independently of GSK-3 and β-TrCP.

Bottom Line: Wnt-5a is considered a noncanonical Wnt as it does not signal by stabilizing beta-catenin in many biological systems.We have uncovered a new noncanonical pathway through which Wnt-5a antagonizes the canonical Wnt pathway by promoting the degradation of beta-catenin.This pathway is Siah2 and APC dependent, but GSK-3 and beta-TrCP independent.

View Article: PubMed Central - PubMed

Affiliation: Genetic Disease Research Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892, USA.

ABSTRACT
Wnts are secreted signaling molecules that can transduce their signals through several different pathways. Wnt-5a is considered a noncanonical Wnt as it does not signal by stabilizing beta-catenin in many biological systems. We have uncovered a new noncanonical pathway through which Wnt-5a antagonizes the canonical Wnt pathway by promoting the degradation of beta-catenin. This pathway is Siah2 and APC dependent, but GSK-3 and beta-TrCP independent. Furthermore, we provide evidence that Wnt-5a also acts in vivo to promote beta-catenin degradation in regulating mammalian limb development and possibly in suppressing tumor formation.

Show MeSH
Related in: MedlinePlus