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Wnt signaling plays important roles in embryonic development, tissue differentiation, and cancer. In both normal and malignant tissue, Wnt family members are often expressed combinatorially, although the significance of this is not understood. We recently showed that Wnt11 and Wnt5a are both required for the initiation of embryonic axis formation and that the two proteins physically interact with each other. However, little is known about the mechanism or biological significance of Wnt-Wnt protein interaction. Here we show in three assays, with Xenopus oocytes, mouse L cells, and human embryonic stem cells, that secreted Xenopus Wnt11/5a complexes have more canonical Wnt signaling activity than secreted Wnt11 or Wnt5a acting alone. We demonstrate that the sulfation activity of tyrosylprotein sulfotransferase-1 (TPST-1) is required for Xenopus dorsal axis formation and that O-sulfation of specific tyrosine residues is necessary for the interaction of Wnt11 with Wnt5a and for enhanced canonical signaling activity. These findings demonstrate a novel aspect of Wnt biology-Wnt family member interaction that depends on tyrosyl sulfation.
Figure 2. Cosynthesized and Secreted Wnt11 and 5a Have Enhanced Canonical Signaling Activity(A and B) show Xenopus paracrine assays; (C and D) show mouse L cells; (E and F) show human ES (hES) cells.(A) Leftmost image: example of a paracrine assay where a dishevelled-GFP mRNA (Dvl2-GFP)-expressing Xenopus blastula animal cap was cocultured for 1 hr with an oocyte injected with either Wnt11-HA mRNA (1 ng), Wnt5a-Myc mRNA (1 ng), or both (500 pg + 500 pg). Middle four images: exogenous Dvl2-GFP fluorescence was visualized in live animal caps stimulated with control uninjected oocytes (Uninj.) or with oocytes secreting Wnt11, Wnt5a, or both proteins. Rightmost four images: Dvl2-GFP was immunostained with anti-GFP antibody in fixed animal caps treated as above.(B) Quantitation of Dvl2-GFP immunostaining measured across the cell membranes in animal caps. The results are means ± SD from three independent experiments.(C) Immunostaining of fixed untreated mouse L cells; L cells treated with conditioned medium only [CM(blank)]; L cells treated with media conditioned by oocytes secreting Wnt5a, Wnt11, or both (Wnt11/5a); or L cells treated with a mixture of Wnt11 and 5a conditioned media (Wnt11+5a) stained with anti-β-catenin antibody (red) and phalloidin (green) for F-actin. Scale bar represents 50 μm.(D) Upper panel: expression levels of cytosolic and total β-catenin protein measured by western blot of lysates of cells from (C). Lower panel: quantitation of cytosolic versus total β-catenin for this experiment. β-tubulin antibody was monitored as a loading control. The experiment was repeated with similar results.(E) Immunostaining of fixed hES cells treated with conditioned medium from oocytes expressing Wnt11-HA mRNA, Wnt5a-Myc mRNA, both (Wnt11/5a), or a mixture of conditioned media from Wnt11-HA- and Wnt5a-Myc-expressing oocytes (Wnt11+5a) stained with anti-β-catenin antibody (green) and DAPI to visualize nuclei (red). Scale bar represents 100 μm.(F) Real-time RT-PCR of hES cells treated as in (E) for 24 hr and analyzed for relative expression levels of Brachyury (Bra). The experiment was repeated three times with similar results. The results are means ± SD from three independent experiments.
