ABSTRACT
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.
Subject(s)
Carcinogenesis/metabolism , Receptors, Wnt/metabolism , Ubiquitin-Protein Ligases/metabolism , Animals , Carcinogenesis/genetics , Humans , Mice , Mice, Inbred BALB C , Oncogene Protein p21(ras)/genetics , Oncogene Protein p21(ras)/metabolism , Phosphorylation , Proteolysis , Receptors, Wnt/genetics , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , Ubiquitin-Protein Ligases/genetics , Wnt Signaling PathwayABSTRACT
Wnt signaling pathways are tightly regulated by ubiquitination, and dysregulation of these pathways promotes tumorigenesis. It has been reported that the ubiquitin ligase RNF43 plays an important role in frizzled-dependent regulation of the Wnt/ß-catenin pathway. Here, we show that RNF43 suppresses both Wnt/ß-catenin signaling and noncanonical Wnt signaling by distinct mechanisms. The suppression of Wnt/ß-catenin signaling requires interaction between the extracellular protease-associated (PA) domain and the cysteine-rich domain (CRD) of frizzled and the intracellular RING finger domain of RNF43. In contrast, these N-terminal domains of RNF43 are not required for inhibition of noncanonical Wnt signaling, but interaction between the C-terminal cytoplasmic region of RNF43 and the PDZ domain of dishevelled is essential for this suppression. We further show the mechanism by which missense mutations in the extracellular portion of RNF43 identified in patients with tumors activate Wnt/ß-catenin signaling. Missense mutations of RNF43 change their localization from the endosome to the endoplasmic reticulum (ER), resulting in the failure of frizzled-dependent suppression of Wnt/ß-catenin signaling. However, these mutants retain the ability to suppress noncanonical Wnt signaling, probably due to interaction with dishevelled. RNF43 is also one of the potential target genes of Wnt/ß-catenin signaling. Our results reveal the molecular role of RNF43 and provide an insight into tumorigenesis.
Subject(s)
DNA-Binding Proteins/genetics , Oncogene Proteins/genetics , Signal Transduction/genetics , Wnt Proteins/genetics , Wnt Signaling Pathway/genetics , Cell Line , Cell Line, Tumor , Cytoplasm/genetics , Cytoskeletal Proteins/genetics , Endoplasmic Reticulum/genetics , Endosomes/genetics , Frizzled Receptors/genetics , HCT116 Cells , HEK293 Cells , HeLa Cells , Hep G2 Cells , Humans , MCF-7 Cells , Mutation, Missense/genetics , RING Finger Domains/genetics , Trans-Activators/genetics , Ubiquitin-Protein Ligases , beta Catenin/geneticsABSTRACT
The nuclear factor (NF)-κB family of transcription factors regulates diverse cellular functions, including inflammation, oncogenesis and apoptosis. It was reported that A20 plays a critical role in the termination of NF-κB signaling after activation. Previously, we showed that Ymer interacts and collaborates with A20, followed by degradation of receptor-interacting protein (RIP) and attenuation of NF-κB signaling. Here we show the function of Ymer in regulation of several signaling pathways including NF-κB on the basis of results obtained by using Ymer transgenic (Ymer Tg) mice. Ymer Tg mice exhibited impaired immune responses, including NF-κB and mitogen-activated protein kinase (MAPK) activation, cell proliferation and cytokine production, to tumor necrosis factor (TNF)-α, polyI:C or lipopolysaccharide (LPS) stimulation. Ymer Tg mice were more resistant to LPS-induced septic shock than wild-type mice. Transgene of Ymer inhibited the onset of glomerulonephritis in lpr/lpr mice as an autoimmune disease model. In contrast to the inflammatory immune response to LPS, Fas-mediated cell death was strongly induced in liver cells of Ymer Tg mice in which Ymer is abundantly expressed. These findings suggest that Ymer acts as a regulator downstream of several receptors and that Ymer functions as a positive or negative regulator in a signaling pathway-dependent manner.