Wnt signaling controls a variety of developmental processes. The canonical Wnt/β-catenin pathway functions to stabilize β-catenin, and the noncanonical Wnt/Ca2+ pathway activates Ca2+/calmodulin-dependent protein kinase II (CaMKII). In addition, the Wnt/Ca2+ pathway activated by Wnt-5a antagonizes the Wnt/β-catenin pathway via an unknown mechanism. The mitogen-activated protein kinase (MAPK) pathway composed of TAK1 MAPK kinase kinase and NLK MAPK also negatively regulates the canonical Wnt/β-catenin signaling pathway. Here we show that activation of CaMKII induces stimulation of the TAK1-NLK pathway. Overexpression of Wnt-5a in HEK293 cells activates NLK through TAK1. Furthermore, by using a chimeric receptor (β2AR-Rfz-2) containing the ligand-binding and transmembrane segments from the β2-adrenergic receptor (β2AR) and the cytoplasmic domains from rat Frizzled-2 (Rfz-2), stimulation with the β-adrenergic agonist isoproterenol activates activities of endogenous CaMKII, TAK1, and NLK and inhibits β-catenin-induced transcriptional activation. These results suggest that the TAK1-NLK MAPK cascade is activated by the noncanonical Wnt-5a/Ca2+ pathway and antagonizes canonical Wnt/β-catenin signaling.

␤-Catenin is a key player in the Wnt signaling pathway, and interacts with cofactor T cell factor/lymphoid enhancer factor (TCF/LEF) to generate a transcription activator complex that activates Wnt-induced genes. We previously reported that Nemo-like kinase (NLK) negatively regulates Wnt signaling via phosphorylation of TCF/LEF. To further evaluate the physiological roles of NLK, we performed yeast two-hybrid screening to identify NLK-interacting proteins. From this screen, we isolated a novel RING finger protein that we term NARF (NLK associated RING finger protein). Here, we show that NARF induces the ubiquitylation of TCF/LEF in vitro and in vivo, and functions as an E3 ubiquitin-ligase that specifically cooperates with the E2 conjugating enzyme E2-25K. We found that NLK augmented NARF binding and ubiquitylation of TCF/LEF, and this required NLK kinase activity. The ubiquitylated TCF/LEF was subsequently degraded by the proteasome. Furthermore, NARF inhibited formation of the secondary axis induced by the ectopic expression of ␤-catenin in Xenopus embryos. Collectively, our findings raise the possibility that NARF functions as a novel ubiquitin-ligase to suppress the Wnt-␤-catenin signaling.The Wnt family of signaling proteins constitutes a large group of highly conserved secreted glycoproteins (1). Wnt proteins are pleiotropic factors that play crucial roles in multiple embryonic developmental processes and also play a role in tumorigenesis (1, 2). Wnt proteins initiate signal transduction via their extracellular surface receptor complex, which is composed of Frizzled proteins (Fz) and lipoprotein receptor-related proteins 5 and 6 (LRP-5/6). In the absence of Wnt stimulation, cytoplasmic ␤-catenin is maintained at low levels by the continuous process of ubiquitin-proteasome-mediated degradation involving a scaffold complex of axin, adenomatous polyposis coli, (APC) and active glycogen synthasekinase-3␤ (GSK-3␤). In the canonical pathway of ␤-catenin signal transduction, Wnt signaling relieves this process of proteasome-mediated degradation, and ␤-catenin consequently accumulates in the cytoplasm. ␤-Catenin then translocates into the nucleus and forms a transcriptional unit with the HMG box class T cell factor/lymphoid enhancer factor (TCF/LEF) 3 to activate expression of its target genes.Nemo-like kinase (NLK) was originally isolated as a murine orthologue of the Drosophila Nemo by RT-PCR from embryonic mouse brain mRNA using degenerate primers designed for the conserved kinase domains I, VI, VII, and IX of the extracellular-signal regulated kinase/mitogen-activated protein kinase (ERK/MAPK) family (3). The amino acid sequence of the NLK kinase domain shows 39 -47% identity to both ERK/ MAPK and cyclin-directed kinase 2. The ERK/MAPK family kinases contain a characteristic conserved phosphorylation motif, Thr-X-Tyr, in their kinase domain VIII that is required for activation. However, the corresponding sequence in NLK is Thr-Gln-Glu, which is quite similar to the sequence Thr-HisGlu found in some cyclin-dire...

Transforming growth factor (TGF)-β-activated kinase 1 (TAK1) and Nemo-like kinase (NLK) function in Xenopus, Drosophila, and Caenorhabditis elegans development. Here we report that serine phosphorylation of STAT3 induced by TAK1-NLK cascade is essential for TGF-β-mediated mesoderm induction in Xenopus embryo. Depletion of TAK1, NLK, or STAT3 blocks TGF-β-mediated mesoderm induction. Coexpression of NLK and STAT3 induces mesoderm by a mechanism that requires serine phosphorylation of STAT3. Activin activates NLK, which in turn directly phosphorylates STAT3. Moreover, depletion of either TAK1 or NLK inhibits endogenous serine phosphorylation of STAT3. These results provide the first evidence that TAK1-NLK-STAT3 cascade participates in TGF-β-mediated mesoderm induction.

Background: The Wnt signal transduction pathway regulates various aspects of embryonal development and has been implicated in promoting cancer. Signalling by Wnts leads to the stabilization of cytosolic β β β β-catenin, which then associates with TCF transcription factors to regulate expression of Wnt-target genes. The Wnt pathway is further subject to crossregulation at various levels by other components.

Microarray is a powerful tool for analysing gene expression patterns in genome-wide view and has greatly contributed to our understanding of spatiotemporal embryonic development at the molecular level. Members of FGF (fibroblast growth factor) family play important roles in embryogenesis, e.g. in organogenesis, proliferation, differentiation, cell migration, angiogenesis, and wound healing. To dissect spatiotemporally the versatile roles of FGF during embryogenesis, we profiled gene expression in Xenopus embryo explants treated with SU5402, a chemical inhibitor specific to FGF receptor 1 ( FGFR1), by microarray. We identified 38 genes that were down-regulated and 5 that were up-regulated in response to SU5402 treatment from stage 10.5 -11.5 and confirmed their FGF-dependent transcription with RT-PCR analysis and whole-mount in situ hybridization (WISH ). Among the 43 genes, we identified 26 as encoding novel proteins and investigated their spatial expression pattern by WISH. Genes whose expression patterns were similar to FGFR1 were further analysed to test whether any of them represented functional FGF target molecules. Here, we report two interesting genes: one is a component of the canonical Ras-MAPK pathway, similar to mammalian mig6 (mitogen-inducible gene 6) acting in muscle differentiation; the other, similar to GPCR4 (G-protein coupled receptor 4), is a promising candidate for a gastrulation movement regulator. These results demonstrate that our approach is a promising strategy for scanning the genes that are essential for the regulation of a diverse array of developmental processes.

Background : Wnt signalling plays a critical role in

To dissect the molecular mechanisms underlying convergent extension (CE), a prominent set of cell movements during Xenopus gastrulation, we performed a functional expression screen and identified a GTPase-activating protein for ADP ribosylation factors (ArfGAP), which we termed XGAP. We demonstrated that XGAP is required to confine or restrict the cellular protrusive activity to the mediolateral ends of cells, where XGAP is normally localized, and therefore for the proper intercalation of cells participating in CE. We also demonstrated that a C-terminal conserved domain of XGAP, but not its GAP activity, is required and sufficient for this intracellular localization and function. We further showed that XGAP physically interacts with the known polarity proteins 14-3-33, aPKC, and PAR-6 and directs them to the mediolateral ends of dorsal mesoderm cells during gastrulation. We propose that XGAP controls CE through the restriction and maintenance of partitioning-defective (PAR) proteins in the regions that harbor protrusive activity.

We used cDNA microarray analysis to screen for FGF target genes in Xenopus embryos treated with the FGFR1 inhibitor SU5402, and identified neurotrophin receptor homolog (NRH) as an FGF target. Causing gain of NRH function by NRH mRNA or loss of NRH function using a Morpholino antisense-oligonucleotide (Mo) led to gastrulation defects without affecting mesoderm differentiation. Depletion of NRH by the Mo perturbed the polarization of cells in the dorsal marginal zone (DMZ), thereby inhibiting the intercalation of the cells during convergent extension as well as the filopodia formation on DMZ cells. Deletion analysis showed that the carboxyl-terminal region of NRH, which includes the "death domain," was necessary and sufficient to rescue gastrulation defects and to induce the protrusive cell morphology. Furthermore, we found that the FGF signal was both capable of inducing filopodia in animal cap cells, where they do not normally form, and necessary for filopodia formation in DMZ cells. Finally, we demonstrated that FGF required NRH function to induce normal DMZ cell morphology. This study is the first to identify an in vivo role for FGF in the regulation of cell morphology, and we have linked this function to the control of gastrulation cell movements via NRH.