Phosphorylated PPPSPXS peptides inhibit β-catenin phosphorylation by GSK3 in vitro.

<p><i>A</i>. 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. <i>B</i>. Four-fold serial dilutions of HA, Phos-A, and A-mut peptides were included in the β-catenin phosphorylation assay. <i>C</i>. Four-fold serial dilutions of Phos-A, and 14-3-3BP peptides were included in the β-catenin phosphorylation assay. <i>D</i>. Four-fold serial dilutions of HA, Phos-E, Phos-A, Phos-C, and Phos-D peptides were included in the β-catenin phosphorylation assay. <i>E</i>. The result from D was quantified via Adobe Photoshop. β-catenin phosphorylation assays were performed in the presence of Axin and CK1 as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004926#pone-0004926-g001" target="_blank">Figure 1C</a>. 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.</p

A working model for LRP6 inhibition of β-catenin phosphorylation by the Axin-GSK3 complex.

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004926#s3" target="_blank">Discussion</a> for details.</p

Peptide design according to the PPPSPXS motifs in human LRP6.

<p><i>A</i>. 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. <i>B</i>. 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) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004926#pone.0004926-Tamai2" target="_blank">[34]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004926#pone.0004926-MacDonald1" target="_blank">[37]</a>.</p

The inhibition of β-catenin phosphorylation by phosphorylated PPPSPXS peptides is specific for GSK3 and independent of Axin function.

<p><i>A</i>. Different Axin constructs used in this study, the full length Axin (amino acid 1–863), AxinΔDix (1-773), and Axin(351-701) are shown. <i>B</i>. 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. <i>C</i> and <i>D</i>. 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004926#pone-0004926-g003" target="_blank">Figure 3</a>. 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. <i>E</i>. 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. <i>F</i>. 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.</p

In vitro reconstitution of Axin-dependent and CK1 priming-dependent β-catenin phosphorylation by GSK3.

<p><i>A</i>. 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. <i>B</i>. 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. <i>C</i>. 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.</p

The Phos-A but not the A-mut peptide induces axis duplication and Xnr3 expression in Xenopus embryos.

<p><i>A, B, C, D</i>. 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). <i>E</i>. 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.</p

The phosphorylated PPPSPXS peptide inhibits phosphorylation of glycogen synthase and Tau by GSK3.

<p><i>A</i>. Recombinant GST-tagged mouse glycogen synthase carboxyl-terminal domain (mGS-CTD) was expressed in bacteria, and purified by glutathione agarose resin. <i>B</i>. In vitro reconstitution of GS phosphorylation by CK2 and GSK3. The phosphorylation reaction products were analyzed using an anti-phospho-Ser641 GS antibody. <i>C</i>. 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. <i>D</i>. In vitro reconstitution of Tau phosphorylation by GSK3. The phosphorylation reaction products were analyzed using an anti-phospho-Tau antibody (PHF1). <i>E</i>. Tau phosphorylation was inhibited by Phos-A, but not A-mut. The phosphorylation reaction products were analyzed using an anti-phospho-Tau antibody (PHF1). <i>F</i> and <i>G</i>. 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.</p