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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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Peptide design according to the PPPSPXS motifs in human LRP6.A. Sequence alignment of the five PPPSPXS motifs in human LRP6 and LRP5 by the Cluster V program. The PPPSPXS motifs are highlighted in color and boxed. B. The sequences of synthetic peptides are shown. The PPPSPXS motifs in the peptides are underlined, and phosphorylated Ser/Thr residues are shown in italics. The C or K residue in the parenthesis at the amino terminus of peptides A, C, D, and E was introduced for protein conjugation purposes (during immunization for antibody production) [34], [37].
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pone-0004926-g002: Peptide design according to the PPPSPXS motifs in human LRP6.A. Sequence alignment of the five PPPSPXS motifs in human LRP6 and LRP5 by the Cluster V program. The PPPSPXS motifs are highlighted in color and boxed. B. The sequences of synthetic peptides are shown. The PPPSPXS motifs in the peptides are underlined, and phosphorylated Ser/Thr residues are shown in italics. The C or K residue in the parenthesis at the amino terminus of peptides A, C, D, and E was introduced for protein conjugation purposes (during immunization for antibody production) [34], [37].

Mentions: A single phosphorylated PPPSPXS motif is sufficient to activate β-catenin signaling in vivo [34], [37]. In order to investigate whether these PPPSPXS motifs might regulate β-catenin amino-terminal phosphorylation in our in vitro assay, we employed 4 phosphorylated PPPSPXS peptides corresponding to A, C, D, and E motifs of LRP6 (Figure 2A), referred to as Phos-A, Phos-C, Phos-D, and Phos-E, respectively, in which the two Ser/Thr residues in the PPPSPXS motifs were phosphorylated (Figure 2B). Although motif B behaves similarly to the other PPPSPXS motifs when tested in isolation in mammalian cells, motif B appears to be the least critical one in the wild type LRP6 [37], [43]. We therefore did not synthesize and test motif B. As controls, we also synthesized an HA peptide, a dually phosphorylated 14-3-3 binding peptide (14-3-3BP), and a mutant motif A peptide (A-mut), which harbors alanine replacement of the two phosphorylated Ser/Thr residues (Figure 2B) and which has been shown to be completely inactive in Wnt/β-catenin signaling in vivo [35].


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)

Peptide design according to the PPPSPXS motifs in human LRP6.A. Sequence alignment of the five PPPSPXS motifs in human LRP6 and LRP5 by the Cluster V program. The PPPSPXS motifs are highlighted in color and boxed. B. The sequences of synthetic peptides are shown. The PPPSPXS motifs in the peptides are underlined, and phosphorylated Ser/Thr residues are shown in italics. The C or K residue in the parenthesis at the amino terminus of peptides A, C, D, and E was introduced for protein conjugation purposes (during immunization for antibody production) [34], [37].
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2654145&req=5

pone-0004926-g002: Peptide design according to the PPPSPXS motifs in human LRP6.A. Sequence alignment of the five PPPSPXS motifs in human LRP6 and LRP5 by the Cluster V program. The PPPSPXS motifs are highlighted in color and boxed. B. The sequences of synthetic peptides are shown. The PPPSPXS motifs in the peptides are underlined, and phosphorylated Ser/Thr residues are shown in italics. The C or K residue in the parenthesis at the amino terminus of peptides A, C, D, and E was introduced for protein conjugation purposes (during immunization for antibody production) [34], [37].
Mentions: A single phosphorylated PPPSPXS motif is sufficient to activate β-catenin signaling in vivo [34], [37]. In order to investigate whether these PPPSPXS motifs might regulate β-catenin amino-terminal phosphorylation in our in vitro assay, we employed 4 phosphorylated PPPSPXS peptides corresponding to A, C, D, and E motifs of LRP6 (Figure 2A), referred to as Phos-A, Phos-C, Phos-D, and Phos-E, respectively, in which the two Ser/Thr residues in the PPPSPXS motifs were phosphorylated (Figure 2B). Although motif B behaves similarly to the other PPPSPXS motifs when tested in isolation in mammalian cells, motif B appears to be the least critical one in the wild type LRP6 [37], [43]. We therefore did not synthesize and test motif B. As controls, we also synthesized an HA peptide, a dually phosphorylated 14-3-3 binding peptide (14-3-3BP), and a mutant motif A peptide (A-mut), which harbors alanine replacement of the two phosphorylated Ser/Thr residues (Figure 2B) and which has been shown to be completely inactive in Wnt/β-catenin signaling in vivo [35].

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