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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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The inhibition of β-catenin phosphorylation by phosphorylated PPPSPXS peptides is specific for GSK3 and independent of Axin function.A. Different Axin constructs used in this study, the full length Axin (amino acid 1–863), AxinΔDix (1-773), and Axin(351-701) are shown. B. Purification of the full length Axin, AxinΔDix, and Axin(351-701) proteins. These Flagged tagged Axin and Axin fragments were expressed in HEK293T cells, purified via M2 agarose (Sigma) resin, and eluted by 0.2 mg/ml Flag peptides. C and D. The Phos-A peptide inhibited GSK3 phosphorylation of β-catenin in the presence of the full length Axin, or AxinΔDIX (C), or Axin(351-701) (D). Four-fold serial dilutions of the Phos-A peptide were tested as in Figure 3. The A-mut peptide was added at the concentration equivalent to that of Phos-A without dilution (10 µM). The phosphorylation reaction products were analyzed using an anti-phospho-Ser33/Ser37/Thr41 β-catenin antibody. E. Inhibition of GSK3 phosphorylation of β-catenin by Phos-A was independent of Axin. Four-fold serial dilutions of the Phos-A peptide (10 µM, 2.5 µM, and 0.63 µM) were included in the β-catenin phosphorylation assay in the absence or presence of Axin. The A-mut peptide was added at the concentration equivalent to that of Phos-A without dilution (10 µM). The phosphorylation reaction products were analyzed using an anti-phospho-Ser33/Ser37/Thr41 β-catenin antibody. Note that in order to achieve and visualize β-catenin phosphorylation by GSK3 in the absence of Axin (lanes 1–5), 5-fold excess amount of GSK3 (2.2 µM) was employed compared to that in the presence of Axin (lanes 6–10), and the film was overexposed. F. β-catenin Ser45 phosphorylation by CK1 was not affected by Phos-A, used at 10 µM and 2.5 µM. A-mut was at 10 µM. The phosphorylation reaction products were analyzed using an anti-phospho-Ser33/Ser37/Thr41 β-catenin antibody and an anti-phospho-Ser45 β-catenin antibody.
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pone-0004926-g004: The inhibition of β-catenin phosphorylation by phosphorylated PPPSPXS peptides is specific for GSK3 and independent of Axin function.A. Different Axin constructs used in this study, the full length Axin (amino acid 1–863), AxinΔDix (1-773), and Axin(351-701) are shown. B. Purification of the full length Axin, AxinΔDix, and Axin(351-701) proteins. These Flagged tagged Axin and Axin fragments were expressed in HEK293T cells, purified via M2 agarose (Sigma) resin, and eluted by 0.2 mg/ml Flag peptides. C and D. The Phos-A peptide inhibited GSK3 phosphorylation of β-catenin in the presence of the full length Axin, or AxinΔDIX (C), or Axin(351-701) (D). Four-fold serial dilutions of the Phos-A peptide were tested as in Figure 3. The A-mut peptide was added at the concentration equivalent to that of Phos-A without dilution (10 µM). The phosphorylation reaction products were analyzed using an anti-phospho-Ser33/Ser37/Thr41 β-catenin antibody. E. Inhibition of GSK3 phosphorylation of β-catenin by Phos-A was independent of Axin. Four-fold serial dilutions of the Phos-A peptide (10 µM, 2.5 µM, and 0.63 µM) were included in the β-catenin phosphorylation assay in the absence or presence of Axin. The A-mut peptide was added at the concentration equivalent to that of Phos-A without dilution (10 µM). The phosphorylation reaction products were analyzed using an anti-phospho-Ser33/Ser37/Thr41 β-catenin antibody. Note that in order to achieve and visualize β-catenin phosphorylation by GSK3 in the absence of Axin (lanes 1–5), 5-fold excess amount of GSK3 (2.2 µM) was employed compared to that in the presence of Axin (lanes 6–10), and the film was overexposed. F. β-catenin Ser45 phosphorylation by CK1 was not affected by Phos-A, used at 10 µM and 2.5 µM. A-mut was at 10 µM. The phosphorylation reaction products were analyzed using an anti-phospho-Ser33/Ser37/Thr41 β-catenin antibody and an anti-phospho-Ser45 β-catenin antibody.

Mentions: Since Axin is a scaffolding protein critical for GSK3 phosphorylation of β-catenin and binds to the phosphorylated PPPSPXS motif [34], [35], [37], we considered the possibility whether inhibition of β-catenin phosphorylation by the phosphorylated PPPSPXP motif involves the binding between Axin and the phosphorylated PPPSPXS motif. To this end we first employed an Axin mutant, AxinΔDIX (Figure 4A and 4B), which lacks the so-called DIX domain required for Axin-binding to LRP5/6 [36] and therefore does not associate with phosphorylated LRP6 or the PPPSPXP motif (H. H and X. H., unpublished results). β-catenin phosphorylation by GSK3 in vitro was promoted by AxinΔDIX as effectively as the wild type Axin; but unexpectedly, this reaction was inhibited by Phos-A in a similar dose-dependent manner in the presence of either the wild type Axin or AxinΔDIX (Figure 4C). This result implies that Phos-A inhibition of β-catenin phosphorylation by GSK3 may not involve its binding to Axin. To address this issue further, we tested another Axin fragment, Axin(351-701) (Figure 4A and 4B), which contains only the β-catenin- and GSK3-binding domains of Axin and promote β-catenin phosphorylation by GSK3 [42]. Indeed β-catenin phosphorylation by GSK3 was promoted by Axin(351-701) similarly to that by the wild type Axin, and importantly, was inhibited by Phos-A in a dose-dependent manner regardless of the presence of Axin or Axin(351-701) (Figure 4D). These results suggest that phosphorylated PPPSPXS peptide inhibited β-catenin phosphorylation by GSK3 via a manner that is independent of PPPSPXS-binding to Axin, perhaps by inhibiting GSK3 directly.


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)

The inhibition of β-catenin phosphorylation by phosphorylated PPPSPXS peptides is specific for GSK3 and independent of Axin function.A. Different Axin constructs used in this study, the full length Axin (amino acid 1–863), AxinΔDix (1-773), and Axin(351-701) are shown. B. Purification of the full length Axin, AxinΔDix, and Axin(351-701) proteins. These Flagged tagged Axin and Axin fragments were expressed in HEK293T cells, purified via M2 agarose (Sigma) resin, and eluted by 0.2 mg/ml Flag peptides. C and D. The Phos-A peptide inhibited GSK3 phosphorylation of β-catenin in the presence of the full length Axin, or AxinΔDIX (C), or Axin(351-701) (D). Four-fold serial dilutions of the Phos-A peptide were tested as in Figure 3. The A-mut peptide was added at the concentration equivalent to that of Phos-A without dilution (10 µM). The phosphorylation reaction products were analyzed using an anti-phospho-Ser33/Ser37/Thr41 β-catenin antibody. E. Inhibition of GSK3 phosphorylation of β-catenin by Phos-A was independent of Axin. Four-fold serial dilutions of the Phos-A peptide (10 µM, 2.5 µM, and 0.63 µM) were included in the β-catenin phosphorylation assay in the absence or presence of Axin. The A-mut peptide was added at the concentration equivalent to that of Phos-A without dilution (10 µM). The phosphorylation reaction products were analyzed using an anti-phospho-Ser33/Ser37/Thr41 β-catenin antibody. Note that in order to achieve and visualize β-catenin phosphorylation by GSK3 in the absence of Axin (lanes 1–5), 5-fold excess amount of GSK3 (2.2 µM) was employed compared to that in the presence of Axin (lanes 6–10), and the film was overexposed. F. β-catenin Ser45 phosphorylation by CK1 was not affected by Phos-A, used at 10 µM and 2.5 µM. A-mut was at 10 µM. The phosphorylation reaction products were analyzed using an anti-phospho-Ser33/Ser37/Thr41 β-catenin antibody and an anti-phospho-Ser45 β-catenin antibody.
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getmorefigures.php?uid=PMC2654145&req=5

pone-0004926-g004: The inhibition of β-catenin phosphorylation by phosphorylated PPPSPXS peptides is specific for GSK3 and independent of Axin function.A. Different Axin constructs used in this study, the full length Axin (amino acid 1–863), AxinΔDix (1-773), and Axin(351-701) are shown. B. Purification of the full length Axin, AxinΔDix, and Axin(351-701) proteins. These Flagged tagged Axin and Axin fragments were expressed in HEK293T cells, purified via M2 agarose (Sigma) resin, and eluted by 0.2 mg/ml Flag peptides. C and D. The Phos-A peptide inhibited GSK3 phosphorylation of β-catenin in the presence of the full length Axin, or AxinΔDIX (C), or Axin(351-701) (D). Four-fold serial dilutions of the Phos-A peptide were tested as in Figure 3. The A-mut peptide was added at the concentration equivalent to that of Phos-A without dilution (10 µM). The phosphorylation reaction products were analyzed using an anti-phospho-Ser33/Ser37/Thr41 β-catenin antibody. E. Inhibition of GSK3 phosphorylation of β-catenin by Phos-A was independent of Axin. Four-fold serial dilutions of the Phos-A peptide (10 µM, 2.5 µM, and 0.63 µM) were included in the β-catenin phosphorylation assay in the absence or presence of Axin. The A-mut peptide was added at the concentration equivalent to that of Phos-A without dilution (10 µM). The phosphorylation reaction products were analyzed using an anti-phospho-Ser33/Ser37/Thr41 β-catenin antibody. Note that in order to achieve and visualize β-catenin phosphorylation by GSK3 in the absence of Axin (lanes 1–5), 5-fold excess amount of GSK3 (2.2 µM) was employed compared to that in the presence of Axin (lanes 6–10), and the film was overexposed. F. β-catenin Ser45 phosphorylation by CK1 was not affected by Phos-A, used at 10 µM and 2.5 µM. A-mut was at 10 µM. The phosphorylation reaction products were analyzed using an anti-phospho-Ser33/Ser37/Thr41 β-catenin antibody and an anti-phospho-Ser45 β-catenin antibody.
Mentions: Since Axin is a scaffolding protein critical for GSK3 phosphorylation of β-catenin and binds to the phosphorylated PPPSPXS motif [34], [35], [37], we considered the possibility whether inhibition of β-catenin phosphorylation by the phosphorylated PPPSPXP motif involves the binding between Axin and the phosphorylated PPPSPXS motif. To this end we first employed an Axin mutant, AxinΔDIX (Figure 4A and 4B), which lacks the so-called DIX domain required for Axin-binding to LRP5/6 [36] and therefore does not associate with phosphorylated LRP6 or the PPPSPXP motif (H. H and X. H., unpublished results). β-catenin phosphorylation by GSK3 in vitro was promoted by AxinΔDIX as effectively as the wild type Axin; but unexpectedly, this reaction was inhibited by Phos-A in a similar dose-dependent manner in the presence of either the wild type Axin or AxinΔDIX (Figure 4C). This result implies that Phos-A inhibition of β-catenin phosphorylation by GSK3 may not involve its binding to Axin. To address this issue further, we tested another Axin fragment, Axin(351-701) (Figure 4A and 4B), which contains only the β-catenin- and GSK3-binding domains of Axin and promote β-catenin phosphorylation by GSK3 [42]. Indeed β-catenin phosphorylation by GSK3 was promoted by Axin(351-701) similarly to that by the wild type Axin, and importantly, was inhibited by Phos-A in a dose-dependent manner regardless of the presence of Axin or Axin(351-701) (Figure 4D). These results suggest that phosphorylated PPPSPXS peptide inhibited β-catenin phosphorylation by GSK3 via a manner that is independent of PPPSPXS-binding to Axin, perhaps by inhibiting GSK3 directly.

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