The Wnt signalling pathway regulates many developmental processes through a complex of beta-catenin and the T-cell factor/lymphoid enhancer factor (TCF/LEF) family of high-mobility-group transcription factors. Wnt stabilizes cytosolic beta-catenin, which then binds to TCF and activates gene transcription. This signalling cascade is conserved in vertebrates, Drosophila and Caenorhabditis elegans. In C. elegans, the proteins MOM-4 and LIT-1 regulate Wnt signalling to polarize responding cells during embryogenesis. MOM-4 and LIT-1 are homologous to TAK1 (a kinase activated by transforming growth factor-beta) mitogen-activated protein-kinase-kinase kinase (MAP3K) and MAP kinase (MAPK)-related NEMO-like kinase (NLK), respectively, in mammalian cells. These results raise the possibility that TAK1 and NLK are also involved in Wnt signalling in mammalian cells. Here we show that TAK1 activation stimulates NLK activity and downregulates transcriptional activation mediated by beta-catenin and TCF. Injection of NLK suppresses the induction of axis duplication by microinjected beta-catenin in Xenopus embryos. NLK phosphorylates TCF/LEF factors and inhibits the interaction of the beta-catenin-TCF complex with DNA. Thus, the TAK1-NLK-MAPK-like pathway negatively regulates the Wnt signalling pathway.

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.

The cytokines IL-1 and TNF induce expression of a series of genes that regulate in¯ammation through activation of NF-kB signal transduction pathways. TAK1, a MAPKKK, is critical for both IL-1-and TNF-induced activation of the NF-kB pathway. TAB2, a TAK1-binding protein, is involved in IL-1-induced NF-kB activation by physically linking TAK1 to TRAF6. However, IL-1-induced activation of NF-kB is not impaired in TAB2-de®cient embryonic ®bro-blasts. Here we report the identi®cation and characterization of a novel protein designated TAB3, a TAB2-like molecule that associates with TAK1 and can activate NF-kB similar to TAB2. Endogenous TAB3 interacts with TRAF6 and TRAF2 in an IL-1-and a TNF-dependent manner, respectively. Furthermore, IL-1 signaling leads to the ubiquitination of TAB2 and TAB3 through TRAF6. Cotransfection of siRNAs directed against both TAB2 and TAB3 inhibit both IL-1-and TNF-induced activation of TAK1 and NF-kB. These results suggest that TAB2 and TAB3 function redundantly as mediators of TAK1 activation in IL-1 and TNF signal transduction.

The signalling protein Wnt regulates transcription factors containing high-mobility-group (HMG) domains to direct decisions on cell fate during animal development. In Caenorhabditis elegans, the HMG-domain-containing repressor POP-1 distinguishes the fates of anterior daughter cells from their posterior sisters throughout development, and Wnt signalling downregulates POP-1 activity in one posterior daughter cell called E. Here we show that the genes mom-4 and lit-1 are also required to downregulate POP-1, not only in E but also in other posterior daughter cells. Consistent with action in a common pathway, mom-4 and lit-1 exhibit similar mutant phenotypes and encode components of the mitogen-activated protein kinase (MAPK) pathway that are homologous to vertebrate transforming-growth-factor-beta-activated kinase (TAK1) and NEMO-like kinase (NLK), respectively. Furthermore, MOM-4 and TAK1 bind related proteins that promote their kinase activities. We conclude that a MAPK-related pathway cooperates with Wnt signal transduction to downregulate POP-1 activity. These functions are likely to be conserved in vertebrates, as TAK1 and NLK can downregulate HMG-domain-containing proteins related to POP-1.

The Wnt/β-catenin signaling pathway regulates many developmental processes by modulating gene expression. Wnt signaling induces the stabilization of cytosolic β-catenin, which then associates with lymphoid enhancer factor and T-cell factor (LEF-1/TCF) to form a transcription complex that activates Wnt target genes. Previously, we have shown that a specific mitogen-activated protein (MAP) kinase pathway involving the MAP kinase kinase kinase TAK1 and MAP kinase-related Nemo-like kinase (NLK) suppresses Wnt signaling. In this study, we investigated the relationships among NLK, β-catenin, and LEF-1/TCF. We found that NLK interacts directly with LEF-1/TCF and indirectly with β-catenin via LEF-1/TCF to form a complex. NLK phosphorylates LEF-1/TCF on two serine/threonine residues located in its central region. Mutation of both residues to alanine enhanced LEF-1 transcriptional activity and rendered it resistant to inhibition by NLK. Phosphorylation of TCF-4 by NLK inhibited DNA binding by the β-catenin-TCF-4 complex. However, this inhibition was abrogated when a mutant form of TCF-4 was used in which both threonines were replaced with valines. These results suggest that NLK phosphorylation on these sites contributes to the down-regulation of LEF-1/TCF transcriptional activity.

[Keywords: Myb; HIPK2; NLK; TAK1; phosphorylation; degradation] Supplemental material is available at http://www.genesdev.org.

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Methylation of histone H3 Lys 9 and Lys 27 (H3K9 and H3K27) is associated with transcriptional silencing. Here we show that KDM7, a JmjC domain-containing protein, catalyzes demethylation of both mono-or dimethylated H3K9 and H3K27. Inhibition of KDM7 orthologs in zebrafish resulted in developmental brain defects. KDM7 interacts with the follistatin gene locus, and KDM7 depletion in mammalian neuronal cells suppressed follistatin gene transcription in association with increased levels of dimethylated H3K9 and H3K27. Our findings identify KDM7 as a dual demethylase for H3K9 and H3K27 that functions as an eraser of silencing marks on chromatin during brain development.Supplemental material is available at http://www.genesdev.org.