NARF, an Nemo-like Kinase (NLK)-associated Ring Finger Protein Regulates the Ubiquitylation and Degradation of T Cell Factor/Lymphoid Enhancer Factor (TCF/LEF)

Abstract

␤-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...

Role of the Wnt/β-catenin pathway in gastric cancer: An in-depth literature review

“…It is thought that CagA promotes the phosphorylation of MUC1, which is a glycoprotein expressed on the apical borders of secretory epithelial cells, thereby facilitating its interaction with b-catenin, and the subsequent transcriptional activation of cyclin D1 [225] . In addition, gene expression profile analyzed in H. pylori-infected and uninfected patients undergoing chronic superficial gastritis showed that the E3 ubiquitin-protein ligase RNF138, which is involved in the ubiquitination and degradation of TCF/LEF, was downregulated by H. pylori [226,227] . Among the multiple ways in which H. pylori can induce Wnt signaling, the bacteria also induces phosphorylation of the Wnt co-receptor LRP6 in a T4SS-dependent manner and involving Dvl-2 and Dvl-3 proteins [228] .…”

Ubiquitin Ligase RNF138 Promotes Episodic Ataxia Type 2-Associated Aberrant Degradation of Human Cav2.1 (P/Q-Type) Calcium Channels

“…Importantly, we identified RNF138 as a potential binding partner of Ca V 2.1. RNF138, also known as NARF, contains a RING finger domain near its N terminus (Anderson et al, 2010) and was demonstrated to be an E3 ubiquitin ligase negatively regulating the Wnt-␤-catenin signaling pathway in Xenopus embryos (Yamada et al, 2006). In neurons, RNF138 may serve as a binding partner of the small integrin-binding ligand osteopontin (Long et al, 2012).…”

Binary cell fate specification duringC. elegansembryogenesis driven by reiterated reciprocal asymmetry of TCF POP-1 and its coactivatorβ-catenin SYS-1

“…However, whereas regulation of ␤-catenin levels by Wnt is well documented in vertebrates and Drosophila, possible regulation of TCF levels by the MAPK pathway is less well documented. A recent report that NLK promotes TCF degradation by facilitating its interaction with an E3 ligase (Yamada et al, 2006), suggests that simultaneous regulation of ␤-catenin and TCF levels in opposite directions might not be unique to the C. elegans embryo.The two ␤-catenins, WRM-1 and SYS-1 It has been suggested that the cellular functions carried out by a single ␤-catenin in vertebrates and flies are carried out by multiple ␤-catenins in worms (Korswagen et al, 2000). ␤-catenin has been shown to undergo importin-independent nuclear import, perhaps by directly binding to the nuclear pore complex through its armadillo repeats, which resemble the heat repeats of importin (Fagotto et al, 1998).…”