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.