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Inhibition of GSK3 phosphorylation of beta-catenin via phosphorylated PPPSPXS motifs of Wnt coreceptor LRP6.

Wu G, Huang H, Garcia Abreu J, He X - PLoS ONE (2009)

Bottom Line: On activation by Wnt, the Wnt co-receptor LDL receptor related protein 6 (LRP6) is phosphorylated at multiple conserved intracellular PPPSPXS motifs by glycogen synthase kinase 3 (GSK3) and casein kinase 1 (CK1), resulting in recruitment of the scaffolding protein Axin to LRP6.We also show that a phosphorylated PPPSPXS peptide is able to activate Wnt/beta-catenin signaling and to induce axis duplication in Xenopus embryos, presumably by inhibition of GSK3 in vivo.Based on these observations, we propose a working model that Axin recruitment to the phosphorylated LRP6 places GSK3 in the vicinity of multiple phosphorylated PPPSPXS motifs, which directly inhibit GSK3 phosphorylation of beta-catenin.

View Article: PubMed Central - PubMed

Affiliation: F M Kirby Neurobiology Center, Children's Hospital Boston, Harvard Medical School, Boston, MA, USA. geng.wu@sjtu.edu.cn

ABSTRACT
The Wnt/beta-catenin signaling pathway plays essential roles in cell proliferation and differentiation, and deregulated beta-catenin protein levels lead to many types of human cancers. On activation by Wnt, the Wnt co-receptor LDL receptor related protein 6 (LRP6) is phosphorylated at multiple conserved intracellular PPPSPXS motifs by glycogen synthase kinase 3 (GSK3) and casein kinase 1 (CK1), resulting in recruitment of the scaffolding protein Axin to LRP6. As a result, beta-catenin phosphorylation by GSK3 is inhibited and beta-catenin protein is stabilized. However, how LRP6 phosphorylation and the ensuing LRP6-Axin interaction lead to the inhibition of beta-catenin phosphorylation by GSK3 is not fully understood. In this study, we reconstituted Axin-dependent beta-catenin phosphorylation by GSK3 and CK1 in vitro using recombinant proteins, and found that the phosphorylated PPPSPXS peptides directly inhibit beta-catenin phosphorylation by GSK3 in a sequence and phosphorylation-dependent manner. This inhibitory effect of phosphorylated PPPSPXS motifs is direct and specific for GSK3 phosphorylation of beta-catenin at Ser33/Ser37/Thr41 but not for CK1 phosphorylation of beta-catenin at Ser45, and is independent of Axin function. We also show that a phosphorylated PPPSPXS peptide is able to activate Wnt/beta-catenin signaling and to induce axis duplication in Xenopus embryos, presumably by inhibition of GSK3 in vivo. Based on these observations, we propose a working model that Axin recruitment to the phosphorylated LRP6 places GSK3 in the vicinity of multiple phosphorylated PPPSPXS motifs, which directly inhibit GSK3 phosphorylation of beta-catenin. This model provides a possible mechanism to account, in part, for inhibition of beta-catenin phosphorylation by Wnt-activated LRP6.

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Phosphorylated PPPSPXS peptides inhibit β-catenin phosphorylation by GSK3 in vitro.A. The HA, Phos-E, Phos-C, Phos-D and Phos-A peptides (left panel) and the HA, Phos-A, and A-mut peptides (right panel) were included in the β-catenin phosphorylation assay. Each peptide was at 10 µM final concentration. B. Four-fold serial dilutions of HA, Phos-A, and A-mut peptides were included in the β-catenin phosphorylation assay. C. Four-fold serial dilutions of Phos-A, and 14-3-3BP peptides were included in the β-catenin phosphorylation assay. D. Four-fold serial dilutions of HA, Phos-E, Phos-A, Phos-C, and Phos-D peptides were included in the β-catenin phosphorylation assay. E. The result from D was quantified via Adobe Photoshop. β-catenin phosphorylation assays were performed in the presence of Axin and CK1 as in Figure 1C. Each peptide was at 10 µM, 2.5 µM, 0.63 µM, and 0.16 µM (four-fold serial dilutions) final concentration. The phosphorylation reaction products were analyzed by western blotting using an anti-phospho-Ser33/Ser37/Thr41 β-catenin antibody and an anti-β-catenin antibody.
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pone-0004926-g003: Phosphorylated PPPSPXS peptides inhibit β-catenin phosphorylation by GSK3 in vitro.A. The HA, Phos-E, Phos-C, Phos-D and Phos-A peptides (left panel) and the HA, Phos-A, and A-mut peptides (right panel) were included in the β-catenin phosphorylation assay. Each peptide was at 10 µM final concentration. B. Four-fold serial dilutions of HA, Phos-A, and A-mut peptides were included in the β-catenin phosphorylation assay. C. Four-fold serial dilutions of Phos-A, and 14-3-3BP peptides were included in the β-catenin phosphorylation assay. D. Four-fold serial dilutions of HA, Phos-E, Phos-A, Phos-C, and Phos-D peptides were included in the β-catenin phosphorylation assay. E. The result from D was quantified via Adobe Photoshop. β-catenin phosphorylation assays were performed in the presence of Axin and CK1 as in Figure 1C. Each peptide was at 10 µM, 2.5 µM, 0.63 µM, and 0.16 µM (four-fold serial dilutions) final concentration. The phosphorylation reaction products were analyzed by western blotting using an anti-phospho-Ser33/Ser37/Thr41 β-catenin antibody and an anti-β-catenin antibody.

Mentions: When the phosphorylated PPPSPXS peptides were added to the in vitro β-catenin phosphorylation assay, the Phos-A, Phos-C, as well as Phos-E peptide each inhibited, interestingly, β-catenin phosphorylation at Ser33/Ser37/Thr41 by GSK3 (Figure 3A, left panel). The Phos-D peptide, which is atypical and has a CPPSPXS motif (Figure 2B), inhibited β-catenin phosphorylation by GSK3 to a less degree (Figure 3A, left panel). We note that in cultured cells motif D also exhibits significant less activity than A, C and E motifs [37]. The A-mut peptide failed to inhibit β-catenin phosphorylation (Figure 3A, right panel). As additional controls, neither the HA peptide nor the unrelated and dually phosphorylated peptide, 14-3-3BP, affected β-catenin phosphorylation by GSK3 (Figure 3B and 3C). Furthermore, when the amount of peptides used in the phosphorylation assay was titrated, Phos-A, Phos-C and Phos-E peptides inhibited β-catenin phosphorylation in a dose-dependent manner (Figure 3B, 3D, 3E), whereas Phos-D was significantly less active (Figure 3D, 3E). The HA and A-mut peptides did not inhibit β-catenin phosphorylation at all concentrations tested (Figure 3B, 3D, 3E). We note that the molar concentrations of phospho-PPPSPXS peptides employed in these titration assays were 0.4×, 1.5×, 6×, and 24× of that of GSK3 (see Methods).


Inhibition of GSK3 phosphorylation of beta-catenin via phosphorylated PPPSPXS motifs of Wnt coreceptor LRP6.

Wu G, Huang H, Garcia Abreu J, He X - PLoS ONE (2009)

Phosphorylated PPPSPXS peptides inhibit β-catenin phosphorylation by GSK3 in vitro.A. The HA, Phos-E, Phos-C, Phos-D and Phos-A peptides (left panel) and the HA, Phos-A, and A-mut peptides (right panel) were included in the β-catenin phosphorylation assay. Each peptide was at 10 µM final concentration. B. Four-fold serial dilutions of HA, Phos-A, and A-mut peptides were included in the β-catenin phosphorylation assay. C. Four-fold serial dilutions of Phos-A, and 14-3-3BP peptides were included in the β-catenin phosphorylation assay. D. Four-fold serial dilutions of HA, Phos-E, Phos-A, Phos-C, and Phos-D peptides were included in the β-catenin phosphorylation assay. E. The result from D was quantified via Adobe Photoshop. β-catenin phosphorylation assays were performed in the presence of Axin and CK1 as in Figure 1C. Each peptide was at 10 µM, 2.5 µM, 0.63 µM, and 0.16 µM (four-fold serial dilutions) final concentration. The phosphorylation reaction products were analyzed by western blotting using an anti-phospho-Ser33/Ser37/Thr41 β-catenin antibody and an anti-β-catenin antibody.
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pone-0004926-g003: Phosphorylated PPPSPXS peptides inhibit β-catenin phosphorylation by GSK3 in vitro.A. The HA, Phos-E, Phos-C, Phos-D and Phos-A peptides (left panel) and the HA, Phos-A, and A-mut peptides (right panel) were included in the β-catenin phosphorylation assay. Each peptide was at 10 µM final concentration. B. Four-fold serial dilutions of HA, Phos-A, and A-mut peptides were included in the β-catenin phosphorylation assay. C. Four-fold serial dilutions of Phos-A, and 14-3-3BP peptides were included in the β-catenin phosphorylation assay. D. Four-fold serial dilutions of HA, Phos-E, Phos-A, Phos-C, and Phos-D peptides were included in the β-catenin phosphorylation assay. E. The result from D was quantified via Adobe Photoshop. β-catenin phosphorylation assays were performed in the presence of Axin and CK1 as in Figure 1C. Each peptide was at 10 µM, 2.5 µM, 0.63 µM, and 0.16 µM (four-fold serial dilutions) final concentration. The phosphorylation reaction products were analyzed by western blotting using an anti-phospho-Ser33/Ser37/Thr41 β-catenin antibody and an anti-β-catenin antibody.
Mentions: When the phosphorylated PPPSPXS peptides were added to the in vitro β-catenin phosphorylation assay, the Phos-A, Phos-C, as well as Phos-E peptide each inhibited, interestingly, β-catenin phosphorylation at Ser33/Ser37/Thr41 by GSK3 (Figure 3A, left panel). The Phos-D peptide, which is atypical and has a CPPSPXS motif (Figure 2B), inhibited β-catenin phosphorylation by GSK3 to a less degree (Figure 3A, left panel). We note that in cultured cells motif D also exhibits significant less activity than A, C and E motifs [37]. The A-mut peptide failed to inhibit β-catenin phosphorylation (Figure 3A, right panel). As additional controls, neither the HA peptide nor the unrelated and dually phosphorylated peptide, 14-3-3BP, affected β-catenin phosphorylation by GSK3 (Figure 3B and 3C). Furthermore, when the amount of peptides used in the phosphorylation assay was titrated, Phos-A, Phos-C and Phos-E peptides inhibited β-catenin phosphorylation in a dose-dependent manner (Figure 3B, 3D, 3E), whereas Phos-D was significantly less active (Figure 3D, 3E). The HA and A-mut peptides did not inhibit β-catenin phosphorylation at all concentrations tested (Figure 3B, 3D, 3E). We note that the molar concentrations of phospho-PPPSPXS peptides employed in these titration assays were 0.4×, 1.5×, 6×, and 24× of that of GSK3 (see Methods).

Bottom Line: On activation by Wnt, the Wnt co-receptor LDL receptor related protein 6 (LRP6) is phosphorylated at multiple conserved intracellular PPPSPXS motifs by glycogen synthase kinase 3 (GSK3) and casein kinase 1 (CK1), resulting in recruitment of the scaffolding protein Axin to LRP6.We also show that a phosphorylated PPPSPXS peptide is able to activate Wnt/beta-catenin signaling and to induce axis duplication in Xenopus embryos, presumably by inhibition of GSK3 in vivo.Based on these observations, we propose a working model that Axin recruitment to the phosphorylated LRP6 places GSK3 in the vicinity of multiple phosphorylated PPPSPXS motifs, which directly inhibit GSK3 phosphorylation of beta-catenin.

View Article: PubMed Central - PubMed

Affiliation: F M Kirby Neurobiology Center, Children's Hospital Boston, Harvard Medical School, Boston, MA, USA. geng.wu@sjtu.edu.cn

ABSTRACT
The Wnt/beta-catenin signaling pathway plays essential roles in cell proliferation and differentiation, and deregulated beta-catenin protein levels lead to many types of human cancers. On activation by Wnt, the Wnt co-receptor LDL receptor related protein 6 (LRP6) is phosphorylated at multiple conserved intracellular PPPSPXS motifs by glycogen synthase kinase 3 (GSK3) and casein kinase 1 (CK1), resulting in recruitment of the scaffolding protein Axin to LRP6. As a result, beta-catenin phosphorylation by GSK3 is inhibited and beta-catenin protein is stabilized. However, how LRP6 phosphorylation and the ensuing LRP6-Axin interaction lead to the inhibition of beta-catenin phosphorylation by GSK3 is not fully understood. In this study, we reconstituted Axin-dependent beta-catenin phosphorylation by GSK3 and CK1 in vitro using recombinant proteins, and found that the phosphorylated PPPSPXS peptides directly inhibit beta-catenin phosphorylation by GSK3 in a sequence and phosphorylation-dependent manner. This inhibitory effect of phosphorylated PPPSPXS motifs is direct and specific for GSK3 phosphorylation of beta-catenin at Ser33/Ser37/Thr41 but not for CK1 phosphorylation of beta-catenin at Ser45, and is independent of Axin function. We also show that a phosphorylated PPPSPXS peptide is able to activate Wnt/beta-catenin signaling and to induce axis duplication in Xenopus embryos, presumably by inhibition of GSK3 in vivo. Based on these observations, we propose a working model that Axin recruitment to the phosphorylated LRP6 places GSK3 in the vicinity of multiple phosphorylated PPPSPXS motifs, which directly inhibit GSK3 phosphorylation of beta-catenin. This model provides a possible mechanism to account, in part, for inhibition of beta-catenin phosphorylation by Wnt-activated LRP6.

Show MeSH
Related in: MedlinePlus