A phospho-switch controls RNF43-mediated degradation of Wnt receptors to suppress tumorigenesis.

Funder: Japan Foundation for Applied Enzymology; doi: https://doi.org/10.13039/100008695Funder: Pancreas Research Foundation of Japan Collaborative Research Project Program of the Medical Institute of Bioregulation, Kyushu University, Japan Joint Research Program of the Institute for Molecular and Cellular Regulation, Gunma University, Japan Grant for Joint Research Project of the Research Institute for Microbial Diseases Osaka UniversityFunder: European Research Council (ERC (639050) and the Interpark Bio-Convergence Center Grant Program.Frequent mutation of the tumour suppressor RNF43 is observed in many cancers, particularly colon malignancies. RNF43, an E3 ubiquitin ligase, negatively regulates Wnt signalling by inducing degradation of the Wnt receptor Frizzled. In this study, we discover that RNF43 activity requires phosphorylation at a triplet of conserved serines. This phospho-regulation of RNF43 is required for zebrafish development and growth of mouse intestinal organoids. Cancer-associated mutations that abrogate RNF43 phosphorylation cooperate with active Ras to promote tumorigenesis by abolishing the inhibitory function of RNF43 in Wnt signalling while maintaining its inhibitory function in p53 signalling. Our data suggest that RNF43 mutations cooperate with KRAS mutations to promote multi-step tumorigenesis via the Wnt-Ras-p53 axis in human colon cancers. Lastly, phosphomimetic substitutions of the serine trio restored the tumour suppressive activity of extracellular oncogenic mutants. Therefore, harnessing phospho-regulation of RNF43 might be a potential therapeutic strategy for tumours with RNF43 mutations

Mice lacking Wnt2b are viable and display a postnatal olfactory bulb phenotype

Wnts are secreted glycoproteins that play important roles in embryonic development. Wnt2b is transiently expressed in the primitive streak (PS) during gastrulation and in several organs during organogenesis. To determine the biological function of Wnt2b during mouse development, we established a conditional null allele of Wnt2b. Mice lacking Wnt2b were viable, fertile, and displayed a normal life span, however, the olfactory bulb in adult Wnt2b mutant mice was significantly reduced in length. Our results suggest that Wnt2b primarily plays a supportive role in gastrulation and organogenesis, functioning redundantly with canonical Wnts, such as Wnt2, in numerous tissues

Protection of vincristine-induced neuropathy by WldS expression and the independence of the activity of Nmnat1

The slow Wallerian degeneration protein (WldS), a fusion protein containing amino-terminal E4B and full-length nicotinamide mononucleotide adenylyltransferase 1 (Nmnat1), delays axon degeneration caused by physical damages, toxins and genetic mutations which result in patients being diagnosed with neurodegenerative diseases. It is still controversial whether the suppression of axonal degeneration by WldS is due to Nmnat1 or other portion. We generated WldS or Nmnat1-overexpressing Neuro2A cell lines, in which neuronal differentiation including neurite elongation can be induced by retinoic acid. The overexpression of WldS delayed the neurite degeneration by vincristine, whereas that of Nmnat1 did not delay it much. Taken together, Nmnat1 is considerably weaker than WldS for protection from toxic injury in vitro, suggesting that amino-terminal region of WldS is likely to be more significant for protection from axonal degeneration

TRIM31 interacts with p52Shc and inhibits Src-induced anchorage-independent growth

Tripartite motif-containing protein (TRIM) family proteins are involved in a broad range of biological processes and, consistently, their alterations result in diverse pathological conditions such as genetic diseases, viral infection and cancer development. In this study, we found that one of the TRIM family proteins, TRIM31, is highly expressed in the gastrointestinal tract and interacts with p52Shc, one of the signal transducers. We also found by a binding assay that almost the whole region other than the RING domain is required for the binding to p52Shc but found by pulse-chase analysis that overexpression of TRIM31 does not affect the stability of p52Shc. Moreover, we found that overexpression of TRIM31 suppresses anchorage-independent cell growth induced by the active form of c-Src. These results suggest that TRIM31 attenuates c-Src signaling via p52Shc under anchorage-independent growth conditions and is potentially associated with growth activity of cells in the gastrointestinal tract

Post-translational Wnt receptor regulation : Is the fog slowly clearing?

Wnt signaling plays pivotal roles during our entire lives, from conception to death, through the regulation of morphogenesis in developing embryos and the maintenance of tissue homeostasis in adults. The regulation of Wnt signaling occurs on several levels: at the receptor level on the plasma membrane, at the beta-catenin protein level in the cytoplasm, and through transcriptional regulation in the nucleus. Several recent studies have focused on the mechanisms of Wnt receptor regulation, following the discovery that the Wnt receptor frizzled (Fzd) is a target of the ubiquitin ligases, RNF43 and ZNRF3. RNF43 and ZNRF3 are homologous genes that are mutated in several cancers. The details underlying their mechanism of action continue to unfold, while at the same time raising many new questions. In this review, we discuss advances and controversies in our understanding of Wnt receptor regulation

Establishment of a newly improved detection system for NF-κB activity

The transcription factor nuclear factor-κB (NF-κB) plays roles in apoptosis, inflammation and oncogenesis. It is important for biological and medical research to understand when proteins of interest are activated in cells, leading to the establishment of a luciferase/EGFP assay to monitor the activation of transcription factors. Here, we describe an improved reporter system for NF-κB, the NF-κB-activated transgene (NAT) system that can detect NF-κB signalling with high sensitivity and specificity. The NAT system consists of large copy numbers of NF-κB consensus sequence and a minimal promoter derived from the mouse interleukin-2 (IL-2) gene. Furthermore, we generated NAT systems with stable or unstable luciferase/EGFP proteins. Stable and unstable types of luciferase/EGFP are suitable for analyzing the accumulation of and the real-time activity of NF-κB signal, respectively. Our findings suggest that the NAT system is effective for in vivo imaging of NF-κB signalling using cells or animals

RNF43 interacts with NEDL1 and regulates p53-mediated transcription

The ubiquitin-proteasomal system plays a crucial role in oncogenesis in colorectal tissues. Recent studies have shown that stability of β-catenin, which functions as an oncogene for colorectal cancer, is regulated by ubiquitin-mediated degradation. It has been reported that a putative E3 ubiquitin ligase, RNF43, is highly expressed in human colorectal carcinoma and that RNF43 promotes cell growth. However, the involvement of RNF43 in carcinogenesis has not been fully elucidated. In this study, we found by using yeast two-hybrid screening that RNF43 binds to NEDD-4-like ubiquitin-protein ligase-1 (NEDL1), which enhances pro-apoptotic activity by p53. In addition, we found that RNF43 also interacts with p53 and that RNF43 suppresses transcriptional activity of p53 in H1299 cells and attenuates apoptosis induced by ultraviolet irradiation. These findings suggest that RNF43 is associated with p53-mediated apoptosis in collaboration with NEDL1 in colorectal carcinogenesis