De-Li Shi

Direct Binding of the PDZ Domain of Dishevelled to a Conserved Internal Sequence in the C-Terminal Region of Frizzled.

The cytoplasmic protein Dishevelled (Dvl) and the associated membrane-bound receptor Frizzled (Fz) are essential in canonical and noncanonical Wnt signaling pathways. However, the molecular mechanisms underlying this signaling are not well understood. By using NMR spectroscopy, we determined that an internal sequence of Fz binds to the conventional peptide binding site in the PDZ domain of Dvl; this type of site typically binds to C-terminal binding motifs. The C-terminal region of the Dvl inhibitor Dapper (Dpr) and Frodo bound to the same site. In Xenopus, Dvl binding peptides of Fz and Dpr/Frodo inhibited canonical Wnt signaling and blocked Wnt-induced secondary axis formation in a dose-dependent manner, but did not block noncanonical Wnt signaling mediated by the DEP domain. Together, our results identify a missing molecular connection within the Wnt pathway. Differences in the binding affinity of the Dvl PDZ domain and its binding partners may be important in regulating signal transduction by Dvl.

The C‐terminal cytoplasmic Lys‐Thr‐X‐X‐X‐Trp motif in frizzled receptors mediates Wnt/β‐catenin signalling

Frizzled receptors are components of the Wnt signalling pathway, but how they activate the canonical Wnt/β‐catenin pathway is not clear. Here we use three distinct vertebrate frizzled receptors (Xfz3, Xfz4 and Xfz7) and describe whether and how their C‐terminal cytoplasmic regions transduce the Wnt/β‐catenin signal. We show that Xfz3 activates this pathway in the absence of exogenous ligands, while Xfz4 and Xfz7 interact with Xwnt5A to activate this pathway. Analysis using chimeric receptors reveals that their C‐terminal cytoplasmic regions are functionally equivalent in Wnt/β‐catenin signalling. Furthermore, a conserved motif (Lys‐Thr‐X‐X‐X‐Trp) located two amino acids after the seventh transmembrane domain is required for activation of the Wnt/β‐catenin pathway and for membrane relocalization and phosphorylation of Dishevelled. Frizzled receptors with point mutations affecting either of the three conserved residues are defective in Wnt/β‐catenin signalling. These findings provide functional evidence supporting a role of this conserved motif in the modulation of Wnt signalling. They are consistent with the genetic features exhibited by Drosophila Dfz3 and Caenorhabditis elegans mom‐5 in which the tryptophan is substituted by a tyrosine.

Frizzled receptor dimerization is sufficient to activate the Wnt/β-catenin pathway

Wnt signaling has an important role in cell-fate determination, tissue patterning and tumorigenesis. Wnt proteins signal through seven-pass transmembrane receptors of the frizzled family to activateβ -catenin-dependent transcription of target genes. Using early Xenopus embryos, we show that frizzled receptors can dimerize and that dimerization is correlated with activation of the Wnt/β-catenin pathway. Co-immunoprecipitation studies revealed that the receptor Xfz3 exists as a dimer when expressed in Xenopus embryos, and it has been shown to activate the Wnt/β-catenin pathway as revealed by expression of the target gene siamois. Xfz3 dimerization requires intramolecular and/or intermolecular disulfide linkages, and the N-terminal extracellular region of the receptor, including the cysteine-rich domain (CRD), is sufficient for dimerization. The receptor Xfz7 behaves differently from Xfz3 when overexpressed in the embryo as Xfz7 is monomeric and is unable to directly activate the Wnt/β-catenin pathway. However, activation of this pathway can be achieved by artificially forcing Xfz7 dimerization. These results provide the first direct evidence for the dimerization of frizzled receptors and suggest that dimerization contributes to transducing the Wnt/β-catenin signal.

Expression of Xfz3, a Xenopus frizzled family member, is restricted to the early nervous system

Recent advances in analyzing wnt signaling have provided evidence that frizzled proteins can function as wnt receptors. We have identified Xfz3, a Xenopus frizzled family member. The amino acid sequence is 89% identical to the product of the murine gene Mfz3, and is predicted to be a serpentine receptor with seven transmembrane domains. Xfz3 is a maternal mRNA with low levels of expression until the end of gastrulation. The expression level increases significantly from neurulation onward. Whole-mount in situ hybridization analysis shows that expression of Xfz3 is highly restricted to the central nervous system. High levels of expression are detected in the anterior neural folds. Low levels of expression are also detected in the optic and otic vesicles, as well as in the pronephros anlage. In addition, Xfz3 mRNA is concentrated in a large band in the midbrain. Overexpression of Xfz3 blocks neural tube closure, resulting in embryos with either bent and strongly reduced anteroposterior axis in a dose-dependent manner. However, it does not affect gastrulation, the expression and localization of organizer-specific genes such as goosecoid, chordin and noggin. Therefore, Xfz3 is not involved in early mesodermal patterning. Injection of RNA encoding GFP-tagged Xfz3 shows that overexpressed proteins can be detected on the cell surface until at least late neurula stage, suggesting that they can exert an effect after gastrulation. Our expression data and functional analyses suggest that the Xfz3 gene product has an antagonizing activity in the morphogenesis during Xenopus development.

Exogenous tenascin inhibits mesodermal cell migration during amphibian gastrulation.

We have used amphibian gastrulation as a model system to study the action of the extracellular matrix (ECM) glycoprotein tenascin on mesodermal cell migration. Tenascin function was assayed in vitro during spreading of isolated cells from the dorsal marginal zone (DMZ) and during cell migration from DMZ explants. Plastic coated with bovine fibronectin or gastrula ECM was used as a substratum. In both cases, tenascin added to the medium inhibited spreading and migration of mesodermal cells. In addition, a substratum coated with a mixture of fibronectin and tenascin was found to prevent mesodermal cell migration. Tenascin was also microinjected into the blastocoel cavity of living embryos at the late blastula stage. This led to a complete arrest of gastrulation in more than 80% of the cases. Scanning electron microscopy of fractures from arrested gastrulae showed that mesodermal cell migration was blocked. Similar injection experiments carried out at the middle gastrula stage demonstrated that tenascin is able to inhibit cell migration after cells have already contacted the ECM. Mesodermal cell migration in the presence of tenascin could be restored in vitro and in vivo by the monoclonal antibody mAb Tn68 which is known to mask a cell binding site of the molecule. Finally, tenascin microinjected into the blastocoel of blastula or gastrula stage embryos bound within 15 min to the ECM fibrils at all the stages studied. Our results show that exogenous tenascin can be incorporated into embryonic ECM and interferes in vivo with the interactions of cells with a fibronectin-rich matrix.

Sulindac Inhibits Canonical Wnt Signaling by Blocking the PDZ Domain of the Protein Dishevelled

The protective anti-cancer effect of nonsteroidal anti-inflammatory drugs (NSAIDs) has attracted much attention, and clinical trials of several NSAIDs are under way for treatment or prevention of various cancers.[1] As NSAIDs are best known for their inhibition of cyclooxygenase 1 and 2 (COX-1/2), they are hypothesized to suppress tumor growth by blocking prostaglandin synthesis.[2] However, this hypothesis does not explain all of the available data.[3–5] Accumulated evidence suggests that some NSAIDs also target the Wnt/ β-catenin signaling pathway in human cancer cells.[4,5a–c] For example, sulindac (Clinoril) has been shown to suppress canonical β-catenin–related Wnt signaling in breast cancer, lung cancer, and colon cancer cell lines.[4e] However, the molecular mechanism of this effect is not clear. Wnt signaling plays crucial roles in embryonic development and in tissue maintenance in adults.[5] Abnormal activation of Wnt signaling is observed in several types of cancers.[5,6] Dishevelled (Dvl) is a key molecule in the Wnt pathways that, through its PDZ domain, relays Wnt signals from membrane-bound Wnt receptors to downstream components.[5,7] We and others have worked to develop small-molecule inhibitors of Dvl PDZ proteinprotein interaction for use in elucidating biological processes and as potential cancer treatment and prevention agents.[8] Here we show that both sulindac and sulindac sulfone bind to the PDZ domain of Dvl and sulindac suppresses Wnt3A-induced β-catenin signaling at the level of Dvl. Our results suggest that the anticancer protective effect of sulindac (and its metabolite) reflect not only COX-1/2 inhibition but also the inhibition of abnormal canonical Wnt signaling via blockade of the Dvl PDZ domain. To test the binding of sulindac and sulindac sulfone to the Dvl PDZ domain, we conducted chemical shift perturbation experiments with nuclear magnetic resonance (NMR) spectroscopy; this method is widely used to characterize protein-ligand interactions.[9] When added to a solution of 15 N-labeled Dvl PDZ domain, both compounds generated chemical shift perturbations that indicated binding to the same region of the Dvl PDZ domain (Figures 1a and S1). The structure of the Dvl PDZ domain comprises six β-strands (βA–βF) and two α-helices (αA and αB). The chemical shift perturbations induced by

Murine frizzled-1 behaves as an antagonist of the canonical Wnt/β-catenin signaling

Activation of the Wnt signaling cascade provides key signals during development and in disease. Wnt signals are transduced by seven-transmembrane Frizzleds (Fzs) and the single transmembrane low density lipoprotein receptor-related proteins 5 or 6. In the course of the analysis of genes regulated by bone morphogenetic protein 2 in mesenchymal cells we found a significant induction of murine Frizzled-1 (mFz1) gene expression. Unexpectedly overexpression of mFz1 dramatically repressed the induction of alkaline phosphatase mediated by either bone morphogenetic protein 2 or Wnt3a in these cells. Moreover mFz1 overexpression significantly repressed both β-catenin translocation into the nucleus and T cell factor signaling mediated by Wnt3a. Importantly microinjection of mFz1 transcript in Xenopus embryo inhibited the ability of Wnt1 to induce the expression of the Wnt/β-catenin target gene Siamois in animal cap assay and secondary axis formation in whole embryo. By using chimeric constructs in which N- and C-terminal segments of mFz1 were replaced by the corresponding parts of Xfz3 we demonstrated that the antagonistic activity resides in the cysteine-rich domain of the N-terminal part. The antagonist activity of mFz1 could be prevented by overexpression of Gαq-(305-359), which specifically uncouples Gq-coupled receptors, suggesting that Gαq signaling contributes to the inhibition of Wnt/β-catenin pathway by mFz1. This is the first time that a Frizzled receptor has been reported to antagonize Wnt/β-catenin.

Identification of transmembrane protein 88 (TMEM88) as a dishevelled-binding protein

Wnt signaling pathways are involved in embryonic development and adult tissue maintenance and have been implicated in tumorigenesis. Dishevelled (Dvl/Dsh) protein is one of key components in Wnt signaling and plays essential roles in regulating these pathways through protein-protein interactions. Identifying and characterizing Dvl-binding proteins are key steps toward understanding biological functions. Given that the tripeptide VWV (Val-Trp-Val) binds to the PDZ domain of Dvl, we searched publically available databases to identify proteins containing the VWV motif at the C terminus that could be novel Dvl-binding partners. On the basis of the cellular localization and expression patterns of the candidates, we selected for further study the TMEM88 (target protein transmembrane 88), a two-transmembrane-type protein. The interaction between the PDZ domain of Dvl and the C-terminal tail of TMEM88 was confirmed by using NMR and fluorescence spectroscopy. Furthermore, in HEK293 cells, TMEM88 attenuated the Wnt/β-catenin signaling induced by Wnt-1 ligand in a dose-dependent manner, and TMEM88 knockdown by RNAi increased Wnt activity. In Xenopus, TMEM88 protein is sublocalized at the cell membrane and inhibits Wnt signaling induced by Xdsh but not β-catenin. In addition, TMEM88 protein inhibits the formation of a secondary axis normally induced by Xdsh. The findings suggest that TMEM88 plays a role in regulating Wnt signaling. Indeed, analysis of microarray data revealed that the expression of the Tmem88 gene was strongly correlated with that of Wnt signaling-related genes in embryonic mouse intestines. Together, we propose that TMEM88 associates with Dvl proteins and regulates Wnt signaling in a context-dependent manner.

The RNA-binding protein Seb4/RBM24 is a direct target of MyoD and is required for myogenesis during Xenopus early development

RNA-binding proteins play an important role to post-transcriptionally regulate gene expression. During early development they exhibit temporally and spatially regulated expression pattern. The expression of Xenopus laevis Seb4 gene, also known as RBM24 in other vertebrates, is restricted to the lateral and ventral mesoderm during gastrulation and then localized to the somitic mesoderm, in a similar pattern as XMyoD gene. Using a hormone-inducible form of MyoD to identify potential direct MyoD target genes, we find that Seb4 expression is directly regulated by MyoD at the gastrula stage. We further show that a 0.65kb X. tropicalis RBM24 regulatory region contains multiple E boxes (CANNTG), which are potential binding sites for MyoD and other bHLH proteins. By injecting a RBM24 reporter construct into the animal pole of X. laevis embryos, we find that this reporter gene is indeed specifically activated by MyoD and repressed by a dominant negative MyoD mutant. Knockdown of Seb4 produces similar effects as those obtained by the dominant negative MyoD mutant, indicating that it is required for the expression of myogenic genes and myogenesis in the embryo. In cultured ectodermal explants, although overexpression of Seb4 has no obvious effect on myogenesis, knockdown of Seb4 inhibits the expression of myogenic genes and myogenesis induced by MyoD. These results reveal that Seb4 is a target of MyoD during myogenesis and is required for myogenic gene expression.

Antagonistic interaction between IGF and Wnt/JNK signaling in convergent extension in Xenopus embryo

The homeobox gene Otx2 is expressed during gastrulation in the anterior domain of the vertebrate embryo and is involved in neural and head induction during Xenopus early development. It also prevents convergent extension movements in trunk and posterior mesoderm. Insulin-like growth factors (IGFs) were shown to have similar function. However, whether they interact and the mechanism by which they affect convergent extension remain unclear. We show that IGF pathway specifically induces the expression of Otx2 in the early gastrula and blocks convergent extension of neuroectoderm and mesoderm through the transcriptional activation of Otx2 gene. Otx2 represses the expression of Xbra and Xwnt-11, and the effects of IGF on gastrulation movements can be partially rescued by antisense Otx2 morpholino oligonucleotide. These indicate that IGF pathway interacts with Otx2 to restrict Xbra and Xwnt-11 expression in the trunk and posterior regions. Consistent with this, we show that inhibition of IGF signaling or Otx2 function induces Xbra and Xwnt11 expression and convergent extension in ectodermal cells. Furthermore, the blockade of convergent extension by IGF-I and Otx2 can be rescued by coexpression of Xwnt-11 or a constitutively active Jun N-terminal kinase (JNK). Because Xbra and Xwnt-11 are required for convergent extension movements and Xwnt-11 activates the non-canonical Wnt-11/JNK pathway, our results reveal a mutually exclusive function between IGF and Wnt-11/JNK pathways in regulating cell behaviours during vertebrate head and trunk development.