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.

Canonical Wnt signaling is mediated by a molecular "switch" that regulates the transcriptional properties of the T-cell factor (TCF) family of DNA-binding proteins. Members of the myeloid translocation gene (MTG) family of transcriptional corepressors are frequently disrupted by chromosomal translocations in acute myeloid leukemia, whereas MTG16 may be inactivated in up to 40% of breast cancer and MTG8 is a candidate cancer gene in colorectal carcinoma. Genetic studies imply that this corepressor family may function in stem cells. Given that mice lacking Myeloid Translocation Gene Related-1 (Mtgr1) fail to maintain the secretory lineage in the small intestine, we surveyed transcription factors that might recruit Mtgr1 in intestinal stem cells or progenitor cells and found that MTG family members associate specifically with TCF4. Coexpression of ␤-catenin disrupted the association between these corepressors and TCF4. Furthermore, when expressed in Xenopus embryos, MTG family members inhibited axis formation and impaired the ability of ␤-catenin and XLef-1 to induce axis duplication, indicating that MTG family members act downstream of ␤-catenin. Moreover, we found that c-Myc, a transcriptional target of the Wnt pathway, was overexpressed in the small intestines of mice lacking Mtgr1, thus linking inactivation of Mtgr1 to the activation of a potent oncogene.Canonical Wnt signaling plays a critical regulatory role in development and in stem cell functions and cellular differentiation (41, 43). Wnts initiate a signaling cascade that leads to the nuclear accumulation of ␤-catenin, which associates with T-cell factor 4 (TCF4), releasing the TCF4-associated transcriptional corepressors and recruiting coactivators to stimulate TCF4-dependent transcription. The intestinal epithelium has been a rich source of information about this pathway. For example, Tcf4 is required for small intestinal stem cell selfrenewal; mice lacking this transcription factor exhaust the capacity for continued replenishment of the epithelium in utero (24). Conversely, hyperactive Wnt signaling is closely associated with colorectal carcinoma, most commonly via inactivation of the APC tumor suppressor, which regulates the levels of ␤-catenin (41, 43). At the end point of the Wnt signaling cascade, ␤-catenin opposes the action of transcriptional corepressors for binding to TCFs to regulate genes that affect cell fate decisions.Two members of the myeloid translocation gene (MTG) family of transcriptional corepressors, MTG on chromosome 8 (MTG8; also known as ETO or RUNX1T1) and MTG on chromosome 16 (MTG16; also known as ETO2 or CBFA2T3), are disrupted by chromosomal translocations in acute myeloid leukemia (10, 29), which suggests that these factors are key regulators of cellular proliferation or differentiation. As would be expected of master regulatory factors, these targets of chromosomal translocations in acute leukemia are commonly mutated in other tumor types as well. A genomewide screen to detect mutations of genes in colorectal and breast can...

Evidence from Drosophila and cultured cell studies support a role for heterotrimeric G proteins in Wnt signaling. Wnt inhibits the degradation of the transcriptional regulator β-catenin. We screened the α subunits of major families of recombinant G protein subunits and Gβγ subunits in a Xenopus egg extract system that reconstitutes β-catenin degradation. We found that Gαo, Gαq, Gαi2, and Gβγ inhibited β-catenin degradation. Gβ1γ2 promoted phosphorylation and activation of the Wnt co-receptor low density lipoprotein receptor-related protein 6 (LRP6) by recruiting synthase kinase 3 (GSK3) to the membrane and enhancing its kinase activity. In both a reporter gene assay and an in vivo assay, c-βARK, an inhibitor of Gβγ, blocked LRP6 activity. Several components of the Wnt/β-catenin pathway formed a complex: Gβ1γ2, LRP6, GSK3, axin, and dishevelled. We propose that heterotrimeric G protein activation results in formation of free Gβγ and Gα, which act cooperatively to inhibit β-catenin degradation and activate β-catenin-mediated transcription.

Although traditional organism-based mutational analysis is powerful in identifying genes involved in specific biological processes, limitations include incomplete coverage and time required for gene identification. Biochemical screens using cell transfection or yeast two-hybrid methods are rapid, but they are limited by cDNA library quality. The recent establishment of "uni-gene sets" has made it feasible to biochemically screen an organism's entire genome. Radiolabeled protein pools prepared from the Drosophila Gene Collection were used in a Drosophila in vitro expression cloning ("DIVEC") screen for substrates of PAN GU kinase, which is crucial for S-M embryonic cell cycles. Ablation of one identified substrate, Mat89Bb, by RNAi produces a polyploid phenotype similar to that of pan gu mutants. Xenopus embryos injected with Mat89Bb morpholinos arrest with polyploid nuclei, and Mat89Bb RNAi in HeLa cells gives rise to multinucleated cells. Thus, Mat89Bb plays an evolutionarily conserved role as a crucial regulator of both cell cycle and development.