Characterization of mouse dishevelled (Dvl) proteins in Wnt/Wingless signaling pathway.

The dishevelled (dsh) gene family encodes cytoplasmic proteins that have been implicated in Wnt/Wingless (Wg) signaling. To demonstrate functional conservation of Dsh family proteins, two mouse homologs of Drosophila Dsh, Dvl-1 and Dvl-2, were biochemically characterized in mouse and Drosophila cell culture systems. We found that treatment with a soluble Wnt-3A leads to hyperphosphorylation of Dvl proteins and a concomitant elevation of the cytoplasmic beta-catenin levels in mouse NIH3T3, L, and C57MG cells. This coincides well with our finding in a Drosophila wing disc cell line, clone-8, that Wg treatment induced hyperphosphorylation of Dsh (Yanagawa, S., van Leeuwen, F., Wodarz, A., Klingensmith, J., and Nusse, R. (1995) Genes Dev. 9, 1087-1097). Furthermore, we showed that mouse Dvl proteins affect downstream components of Drosophila Wg signaling as Dsh does; overexpression of Dvl proteins in clone-8 cells results in elevation of Armadillo (Drosophila homolog of beta-catenin) and Drosophila E-cadherin levels, hyperphosphorylation of Dvl proteins themselves, and inhibition of Zeste-White3 kinase-mediated phosphorylation of a microtubule-binding protein, Tau. In addition, casein kinase II was shown to coimmunoprecipitate with Dvl proteins, and Dvl proteins were phosphorylated in these immune complexes. These results are direct evidence that Dsh family proteins mediate a set of conserved biochemical processes in the Wnt/Wg signaling pathway.     

Control of beta-catenin phosphorylation/degradation by a dual-kinase mechanism

Wnt regulation of beta-catenin degradation is essential for development and carcinogenesis. beta-catenin degradation is initiated upon amino-terminal serine/threonine phosphorylation, which is believed to be performed by glycogen synthase kinase-3 (GSK-3) in complex with tumor suppressor proteins Axin and adnomatous polyposis coli (APC). Here we describe another Axin-associated kinase, whose phosphorylation of beta-catenin precedes and is required for subsequent GSK-3 phosphorylation of beta-catenin. This "priming" kinase is casein kinase Ialpha (CKIalpha). Depletion of CKIalpha inhibits beta-catenin phosphorylation and degradation and causes abnormal embryogenesis associated with excessive Wnt/beta-catenin signaling. Our study uncovers distinct roles and steps of beta-catenin phosphorylation, identifies CKIalpha as a component in Wnt/beta-catenin signaling, and has implications to pathogenesis/therapeutics of human cancers and diabetes.     

Domains of axin involved in protein-protein interactions, Wnt pathway inhibition, and intracellular localization.

Axin was identified as a regulator of embryonic axis induction in vertebrates that inhibits the Wnt signal transduction pathway. Epistasis experiments in frog embryos indicated that Axin functioned downstream of glycogen synthase kinase 3beta (GSK3beta) and upstream of beta-catenin, and subsequent studies showed that Axin is part of a complex including these two proteins and adenomatous polyposis coli (APC). Here, we examine the role of different Axin domains in the effects on axis formation and beta-catenin levels. We find that the regulators of G-protein signaling domain (major APC-binding site) and GSK3beta-binding site are required, whereas the COOH-terminal sequences, including a protein phosphatase 2A binding site and the DIX domain, are not essential. Some forms of Axin lacking the beta-catenin binding site can still interact indirectly with beta-catenin and regulate beta-catenin levels and axis formation. Thus in normal embryonic cells, interaction with APC and GSK3beta is critical for the ability of Axin to regulate signaling via beta-catenin. Myc-tagged Axin is localized in a characteristic pattern of intracellular spots as well as at the plasma membrane. NH2-terminal sequences were required for targeting to either of these sites, whereas COOH-terminal sequences increased localization at the spots. Coexpression of hemagglutinin-tagged Dishevelled (Dsh) revealed strong colocalization with Axin, suggesting that Dsh can interact with the Axin/APC/GSK3/beta-catenin complex, and may thus modulate its activity.     

Inhibition of Wnt signaling pathway by a novel axin-binding protein

Axin forms a complex with adenomatous polyposis coli gene product, glycogen synthase kinase-3beta (GSK-3beta), beta-catenin, Dvl, and protein phosphatase 2A and functions as a scaffold protein in the Wnt signaling pathway. In the Axin complex, GSK-3beta efficiently phosphorylates beta-catenin, which is then ubiquitinated and degraded by proteasome. We isolated a novel protein that binds to Axin and named it Axam (for Axin associating molecule). Axam formed a complex with Axin in intact cells and bound directly to Axin. Axam inhibited the complex formation of Dvl with Axin and the activity of Dvl to suppress GSK-3beta-dependent phosphorylation of Axin. Furthermore, Axam induced the degradation of beta-catenin in SW480 cells and inhibited Wnt-dependent axis duplication in Xenopus embryos. These results suggest that Axam regulates the Wnt signaling pathway negatively by inhibiting the binding of Dvl to Axin.     

Structural basis of the Axin-adenomatous polyposis coli interaction

Axin and the adenomatous polyposis coli (APC) tumor suppressor protein are components of the Wnt/Wingless growth factor signaling pathway. In the absence of Wnt signal, Axin and APC regulate cytoplasmic levels of the proto-oncogene beta-catenin through the formation of a large complex containing these three proteins, glycogen synthase kinase 3beta (GSK3beta) and several other proteins. Both Axin and APC are known to be critical for beta-catenin regulation, and truncations in APC that eliminate the Axin-binding site result in human cancers. A protease-resistant domain of Axin that contains the APC-binding site is a member of the regulators of G-protein signaling (RGS) superfamily. The crystal structures of this domain alone and in complex with an Axin-binding sequence from APC reveal that the Axin-APC interaction occurs at a conserved groove on a face of the protein that is distinct from the G-protein interface of classical RGS proteins. The molecular interactions observed in the Axin-APC complex provide a rationale for the evolutionary conservation seen in both proteins.     

The C-terminal domain of armadillo binds to hypophosphorylated teashirt to modulate wingless signalling in Drosophila

Wnt signalling is a key pathway for tissue patterning during animal development. In Drosophila, the Wnt protein Wingless acts to stabilize Armadillo inside cells where it binds to at least two DNA-binding factors which regulate specific target genes. One Armadillo-binding protein in Drosophila is the zinc finger protein Teashirt. Here we show that Wingless signalling promotes the phosphorylation and the nuclear accumulation of Teashirt. This process requires the binding of Teashirt to the C-terminal end of Armadillo. Finally, we present evidence that the serine/threonine kinase Shaggy is associated with Teashirt in a complex. We discuss these results with respect to current models of Armadillo/beta-catenin action for the transmission of the Wingless/Wnt pathway.     

Interaction among GSK-3, GBP, axin, and APC in Xenopus axis specification

Glycogen synthase kinase 3 (GSK-3) is a constitutively active kinase that negatively regulates its substrates, one of which is beta-catenin, a downstream effector of the Wnt signaling pathway that is required for dorsal-ventral axis specification in the Xenopus embryo. GSK-3 activity is regulated through the opposing activities of multiple proteins. Axin, GSK-3, and beta-catenin form a complex that promotes the GSK-3-mediated phosphorylation and subsequent degradation of beta-catenin. Adenomatous polyposis coli (APC) joins the complex and downregulates beta-catenin in mammalian cells, but its role in Xenopus is less clear. In contrast, GBP, which is required for axis formation in Xenopus, binds and inhibits GSK-3. We show here that GSK-3 binding protein (GBP) inhibits GSK-3, in part, by preventing Axin from binding GSK-3. Similarly, we present evidence that a dominant-negative GSK-3 mutant, which causes the same effects as GBP, keeps endogenous GSK-3 from binding to Axin. We show that GBP also functions by preventing the GSK-3-mediated phosphorylation of a protein substrate without eliminating its catalytic activity. Finally, we show that the previously demonstrated axis-inducing property of overexpressed APC is attributable to its ability to stabilize cytoplasmic beta-catenin levels, demonstrating that APC is impinging upon the canonical Wnt pathway in this model system. These results contribute to our growing understanding of how GSK-3 regulation in the early embryo leads to regional differences in beta-catenin levels and establishment of the dorsal axis.     

CKIepsilon/discs overgrown promotes both Wnt-Fz/beta-catenin and Fz/PCP signaling in Drosophila

The related Wnt-Frizzled(Fz)/beta-catenin and Fz/planar cell polarity (PCP) pathways are essential for the regulation of numerous developmental processes and are deregulated in many human diseases. Both pathways require members of the Dishevelled (Dsh or Dvl) family of cytoplasmic factors for signal transduction downstream of the Fz receptors. Dsh family members have been studied extensively, but their activation and regulation remains largely unknown. In particular, very little is known about how Dsh differentially signals to the two pathways. Recent work in cell culture has suggested that phosphorylation of Dsh by Casein Kinase I epsilon (CKIepsilon) may act as a molecular "switch," promoting Wnt/beta-catenin while inhibiting Fz/PCP signaling. Here, we demonstrate in vivo in Drosophila through a series of loss-of-function and coexpression assays that CKIepsilon acts positively for signaling in both pathways, rather than as a switch. Our data suggest that the kinase activity of CKIepsilon is required for peak levels of Wnt/beta-catenin signaling. In contrast, CKIepsilon is a mandatory signaling factor in the Fz/PCP pathway, possibly through a kinase-independent mechanism. Furthermore, we have identified the primary kinase target residue of CKIepsilon on Dsh. Thus, our data suggest that CKIepsilon modulates Wnt/beta-catenin and Fz/PCP signaling pathways via kinase-dependent and -independent mechanisms.     

An F-box protein, FWD1, mediates ubiquitin-dependent proteolysis of beta-catenin

beta-catenin plays an essential role in the Wingless/Wnt signaling cascade and is a component of the cadherin cell adhesion complex. Deregulation of beta-catenin accumulation as a result of mutations in adenomatous polyposis coli (APC) tumor suppressor protein is believed to initiate colorectal neoplasia. beta-catenin levels are regulated by the ubiquitin-dependent proteolysis system and beta-catenin ubiquitination is preceded by phosphorylation of its N-terminal region by the glycogen synthase kinase-3beta (GSK-3beta)/Axin kinase complex. Here we show that FWD1 (the mouse homologue of Slimb/betaTrCP), an F-box/WD40-repeat protein, specifically formed a multi-molecular complex with beta-catenin, Axin, GSK-3beta and APC. Mutations at the signal-induced phosphorylation site of beta-catenin inhibited its association with FWD1. FWD1 facilitated ubiquitination and promoted degradation of beta-catenin, resulting in reduced cytoplasmic beta-catenin levels. In contrast, a dominant-negative mutant form of FWD1 inhibited the ubiquitination process and stabilized beta-catenin. These results suggest that the Skp1/Cullin/F-box protein FWD1 (SCFFWD1)-ubiquitin ligase complex is involved in beta-catenin ubiquitination and that FWD1 serves as an intracellular receptor for phosphorylated beta-catenin. FWD1 also links the phosphorylation machinery to the ubiquitin-proteasome pathway to ensure prompt and efficient proteolysis of beta-catenin in response to external signals. SCFFWD1 may be critical for tumor development and suppression through regulation of beta-catenin protein stability.