Wu G , Huang H , Garcia Abreu J , He X .

R01-GM74241 NIGMS NIH HHS , R01 GM074241 NIGMS NIH HHS

	Figure 1. In vitro reconstitution of Axin-dependent and CK1 priming-dependent β-catenin phosphorylation by GSK3.A. Recombinant GST-β-catenin, Flag-CK1, MBP-Axin, and His-GSK3 proteins were expressed in bacteria or insect cells and purified by glutathione agarose, anti-Flag M2 agarose, amylose resin, or Ni-NTA resin, respectively. In the case of β-catenin, GST was cleaved via thrombin and purified away from β-catenin. * indicates each recombinant protein. B. β-catenin phosphorylation by CK1 was reconstituted in vitro using purified proteins. The phosphorylation reaction products were analyzed by western blotting using an anti-phospho-Ser45 β-catenin antibody. C. Axin-dependent phosphorylation by GSK3 was reconstituted in vitro using purified proteins. For Axin-dependent β-catenin phosphorylation in this and other figures, 0.43 µM of GSK3, 0.54 µM of CK1α, 0.21 µM of Axin, and 0.73 µM of β-catenin were used in each assay. The phosphorylation reaction products were analyzed by western blotting using an anti-phospho-Ser45 β-catenin antibody and an anti-phospho-Ser33/Ser37/Thr41 β-catenin antibody.
	Figure 2. 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].
	Figure 3. 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.
	Figure 4. 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.
	Figure 5. The phosphorylated PPPSPXS peptide inhibits phosphorylation of glycogen synthase and Tau by GSK3.A. Recombinant GST-tagged mouse glycogen synthase carboxyl-terminal domain (mGS-CTD) was expressed in bacteria, and purified by glutathione agarose resin. B. In vitro reconstitution of GS phosphorylation by CK2 and GSK3. The phosphorylation reaction products were analyzed using an anti-phospho-Ser641 GS antibody. C. GS phosphorylation at Ser641 was inhibited by Phos-A, but not by A-mut. The phosphorylation reaction products were analyzed using an anti-phospho-Ser641 GS antibody. D. In vitro reconstitution of Tau phosphorylation by GSK3. The phosphorylation reaction products were analyzed using an anti-phospho-Tau antibody (PHF1). E. Tau phosphorylation was inhibited by Phos-A, but not A-mut. The phosphorylation reaction products were analyzed using an anti-phospho-Tau antibody (PHF1). F and G. Different concentrations of Phos-A inhibited β-catenin and Tau phosphorylation by GSK3 in a similar manner. A four-fold dilution of Phos-A was employed. The graph represents the average of three independent experiments.
	Figure 6. The Phos-A but not the A-mut peptide induces axis duplication and Xnr3 expression in Xenopus embryos.A, B, C, D. Uninjected embryo (A), A-mut-injected embryo (B), and Phos-A-injected embryo (C) shown at neural fold stage. The duplicated partial axis is labeled by the red arrowhead. Ventrally injected Phos-A (4.8 ng/embryo) induced axis duplication in 19% embryos (10 of 52). A-mut (4.8 ng/embryo) did not induce axis duplication (0 of 60). Three independent experiments were combined (D). E. Phos-A (3 and 4.8 ng/embryo) but not A-mut (4.8 ng/embryo) induced Xnr3 expression in animal pole explants, as assayed by RT-PCR. Xenopus Wnt8 RNA injection (8 pg/embryo) served as a positive control. The activity of the Phos-A peptide was significantly weaker than that of Wnt8 RNA, likely due to dilution, proteolysis and/or dephosphorylation in the embryo in the absence of any de novo synthesis. WE: whole embryo. EF1-a: loading control. –RT: without reverse transcriptase.
	Figure 7. A working model for LRP6 inhibition of β-catenin phosphorylation by the Axin-GSK3 complex.While one of the five phosphorylated PPPSPXS motifs of LRP6 physically interacts with Axin, other phosphorylated PPPSPXS motifs may directly inhibit GSK3 phosphorylation of β-catenin in the Axin complex. Axin-binding to motif C is drawn arbitrarily. See Discussion for details.

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