Recent studies have shown that Drosophila Dishevelled (Dsh), an essential component of the wingless signal transduction, is also involved in planar polarity signaling through the c-Jun N-terminal kinase (JNK)/ stress-activated protein kinase (SAPK) pathway in Drosophila. Here, we show that expression of a mouse homolog of Dsh (mDvl-1) in NIH3T3 cells activates JNK/ SAPK, and its activator MKK7. A C-terminal half of mDvl-1 which contains the DEP domain was sufficient for the activation of JNK/SAPK, whereas an N-terminal half of mDvl-1 as well as the DEP domain is required for stimulation of the TCF/LEF-1-dependent transcriptional activation, a ␤-catenin-dependent process. A single amino acid substitution (Met for Lys) within the DEP domain (mDvl-1 (KM)) abolished the JNK/SAPK-activating activity of mDvl-1, but did not affect the activity to activate the LEF-1-dependent transcription. Ectopic expression of mDvl-1 (KM) or an N-terminal half of mDvl-1, but not the C-terminal, was able to induce secondary axis in Xenopus embryos. Because the secondary axis formation is dependent on the Wnt/␤-catenin signaling pathway, these results suggest that distinct domains of mDvl-1 are responsible for the two downstream signaling pathways, the ␤-catenin pathway and the JNK/SAPK pathway in vertebrates. c-Jun N-terminal kinase (JNK)1 /stress-activated protein kinase (SAPK), a member of mitogen-activated protein kinases (MAPKs) (1-4), is activated by exposure of cells to certain kinds of cytokines and environmental stresses (4 -7). Two MAPK kinases (MAPKKs), MKK4/SEK1 and MKK7, act as direct activators for JNK/SAPK (4 -12). Although several members of MAPKK kinases, Ste20 homologs, protein-tyrosine kinases, and low molecular weight GTPases have been described as upstream components of the JNK/SAPK signaling pathway, players involved in signaling pathways have not been completely listed up, and exact signaling pathways from receptors or sensors to the activation of JNK/SAPK have not been fully understood.The Wnt proteins constitute a family of secreted glycoproteins, among which Wingless (Wg) in Drosophila is the best characterized member. Genetic evidence reveals that Wg signals through an intracellular cascade that includes Dishevelled (Dsh), Zeste-White3/Shaggy and Armadillo, a ␤-catenin homolog (13). There exist vertebrate counterparts of these genes, and the corresponding cascade is believed to function in a variety of biological processes (14, 15). Of the known components, Dsh is thought to act most immediately downstream of the receptor. Alignment of members of invertebrate and vertebrate Dsh family proteins (16 -19) reveals three conserved domains: a DIX domain, a PDZ domain, and a DEP domain (20). Recent analysis of the planar polarity-specific dsh 1 allele in Drosophila revealed a single amino acid point mutation in the DEP domain and demonstrated requirement of the DEP domain for planar polarity signaling but not for wingless signaling (21, 22). Moreover, it has been shown that Dsh may act through low molecular weight GT...

␤-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...

The fibroblast growth factor (FGF)/MAPK pathway plays an important role in early Xenopus developmental processes, including mesoderm patterning. The activation of the MAPK pathway leads to induction of Xenopus Brachyury (Xbra), which regulates the transcription of downstream mesoderm-specific genes in mesoderm patterning. However, the link between the FGF/MAPK pathway and the induction of Xbra has not been fully understood. Here we present evidence suggesting that Ets-2 is involved in the induction of Xbra and thus in the development of posterior mesoderm during early embryonic development. Overexpression of Ets-2 caused posteriorized embryos and led to the induction of mesoderm in ectodermal explants. Expression of a dominant-negative form of Ets-2 or injection of antisense morpholino oligonucleotides against Ets-2 inhibited the formation of the trunk and tail structures. Overexpression of Ets-2 resulted in the induction of Xbra, and expression of the dominant-negative Ets-2 inhibited FGFor constitutively active MEK-induced Xbra expression. Moreover, overexpression of Ets-2 up-regulated the transcription from Xbra promoter reporter gene constructs. Ets-2 bound to the Xbra promoter region in vitro. These results taken together indicate that Xenopus Ets-2 plays an essential role in mesoderm patterning, lying between the FGF/MAPK pathway and the Xbra transcription.