Wnt/β-catenin signaling is critically involved in metazoan development, stem cell maintenance and human disease. Using Xenopus laevis egg extract to screen for compounds that both stabilize Axin and promote β-catenin turnover, we identified an FDA-approved drug, pyrvinium, as a potent inhibitor of Wnt signaling (EC50 of ~10 nM). We show pyrvinium binds all casein kinase 1 (CK1) family members in vitro at low nanomolar concentrations and pyrvinium selectively potentiates casein kinase 1α (CK1α) kinase activity. CK1α knockdown abrogates the effects of pyrvinium on the Wnt pathway. In addition to its effects on Axin and β-catenin levels, pyrvinium promotes degradation of Pygopus, a Wnt transcriptional component. Pyrvinium treatment of colon cancer cells with mutation of the gene for adenomatous polyposis coli (APC) or β-catenin inhibits both Wnt signaling and proliferation. Our findings reveal allosteric activation of CK1α as an effective mechanism to inhibit Wnt signaling and highlight a new strategy for targeted therapeutics directed against the Wnt pathway.

Wnt/␤-catenin signaling controls various cell fates in metazoan development and is misregulated in several cancers and developmental disorders. Binding of a Wnt ligand to its transmembrane coreceptors inhibits phosphorylation and degradation of the transcriptional coactivator ␤-catenin, which then translocates to the nucleus to regulate target gene expression. To understand how Wnt signaling prevents ␤-catenin degradation, we focused on the Wnt coreceptor low-density lipoprotein receptor-related protein 6 (LRP6), which is required for signal transduction and is sufficient to activate Wnt signaling when overexpressed. LRP6 has been proposed to stabilize ␤-catenin by stimulating degradation of Axin, a scaffold protein required for ␤-catenin degradation. In certain systems, however, Wnt-mediated Axin turnover is not detected until after ␤-catenin has been stabilized. Thus, LRP6 may also signal through a mechanism distinct from Axin degradation. To establish a biochemically tractable system to test this hypothesis, we expressed and purified the LRP6 intracellular domain from bacteria and show that it promotes ␤-catenin stabilization and Axin degradation in Xenopus egg extract. Using an Axin mutant that does not degrade in response to LRP6, we demonstrate that LRP6 can stabilize ␤-catenin in the absence of Axin turnover. Through experiments in egg extract and reconstitution with purified proteins, we identify a mechanism whereby LRP6 stabilizes ␤-catenin independently of Axin degradation by directly inhibiting GSK3's phosphorylation of ␤-catenin.Axin ͉ GSK3 ͉ LRP6 ͉ Wnt

and supported by the DFG. We are grateful to Drs Eddy De Robertis, Herbert Steinbeisser, Michael Sargent and Mark Mercola for plasmids. We thank the members of our laboratories for discussions; Hiromasa Ninomiya and Mark Makowiecki for suggestions to improve the manuscript; and Carl-Philipp Heisenberg for communication of a manuscript before publication. ReferencesAndersson, M., Ostman, A., Westermark, B. and Heldin, C.-H. (1994).Characterization of the retention motif in the C-terminal part of the long splice form of platelet-derived growth factor A-chain. (1996). Mutation of a Src phosphorylation site in the PDGF beta-receptor leads to increased PDGF-stimulated chemotaxis but decreased mitogenesis. EMBO J. 15, 5299-5313.

We have undertaken the first detailed analysis of Rho GTPase function during vertebrate development by analyzing how RhoA and Rac1 control convergent extension of axial mesoderm during Xenopus gastrulation. Monitoring of a number of parameters in time-lapse recordings of mesoderm explants revealed that Rac and Rho have both distinct and overlapping roles in regulating the motility of axial mesoderm cells. The cell behaviors revealed by activated or inhibitory versions of these GTPases in native tissue were clearly distinct from those previously documented in cultured fibroblasts. The dynamic properties and polarity of protrusive activity, along with lamellipodia formation, were controlled by the two GTPases operating in a partially redundant manner, while Rho and Rac contributed separately to cell shape and filopodia formation. We propose that Rho and Rac operate in distinct signaling pathways that are integrated to control cell motility during convergent extension.

*Wnt signaling regulates ␤-catenin-mediated gene transcription and planar cell polarity (PCP). The Wnt co-receptor, Lrp6, is required for signaling along the ␤-catenin arm. We show that Lrp6 downregulation (by morpholino injection) or overexpression in Xenopus embryos disrupts convergent extension, a hallmark feature of Wnt/PCP components. In embryos with decreased Lrp6 levels, cells of the dorsal marginal zone (DMZ), which undergoes extensive cellular rearrangements during gastrulation, exhibit decreased length:width ratios, decreased migration, and increased numbers of transient cytoplasmic protrusions. We show that Lrp6 opposes Wnt11 activity and localizes to the posterior edge of migrating DMZ cells and that Lrp6 downregulation enhances cortical and nuclear localization of Dsh and phospho-JNK, respectively. Taken together, these data suggest that Lrp6 inhibits Wnt/PCP signaling. Finally, we identify the region of the Lrp6 protein with Wnt/PCP activity to a stretch of 36 amino acids, distinct from regions required for Wnt/␤-catenin signaling. We propose a model in which Lrp6 plays a critical role in the switch from Wnt/PCP to Wnt/␤-catenin signaling.