scholarly journals RNF43 inhibits WNT5A-driven signaling and suppresses melanoma invasion and resistance to the targeted therapy

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Tomasz Radaszkiewicz ◽  
Michaela Nosková ◽  
Kristína Gömöryová ◽  
Olga Vondálová Blanářová ◽  
Katarzyna Anna Radaszkiewicz ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Wnt Pathway ◽  
Negative Regulator ◽  
Braf V600e ◽  
Receptor Complex ◽  
The Core ◽  
Development Of Resistance ◽  
Biological Responses ◽  
Worse Prognosis

RNF43 is an E3 ubiquitin ligase and known negative regulator of WNT/β-catenin signaling. We demonstrate that RNF43 is also a regulator of noncanonical WNT5A-induced signaling in human cells. Analysis of the RNF43 interactome using BioID and immunoprecipitation showed that RNF43 can interact with the core receptor complex components dedicated to the noncanonical Wnt pathway such as ROR1, ROR2, VANGL1, and VANGL2. RNF43 triggers VANGL2 ubiquitination and proteasomal degradation and clathrin-dependent internalization of ROR1 receptor and inhibits ROR2 activation. These activities of RNF43 are physiologically relevant and block pro-metastatic WNT5A signaling in melanoma. RNF43 inhibits responses to WNT5A, which results in the suppression of invasive properties of melanoma cells. Furthermore, RNF43 prevented WNT5A-assisted development of resistance to BRAF V600E and MEK inhibitors. Next, RNF43 acted as melanoma suppressor and improved response to targeted therapies in vivo. In line with these findings, RNF43 expression decreases during melanoma progression and RNF43-low patients have a worse prognosis. We conclude that RNF43 is a newly discovered negative regulator of WNT5A-mediated biological responses that desensitizes cells to WNT5A.

scholarly journals RNF43 inhibits WNT5A driven signaling and suppresses melanoma invasion

2021 ◽  
Author(s):  
Tomasz Radaszkiewicz ◽  
Michaela Nosková ◽  
Kristína Gömöryová ◽  
Olga Vondálová Blanářová ◽  
Katarzyna Anna Radaszkiewicz ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Wnt Pathway ◽  
E3 Ubiquitin Ligase ◽  
Negative Regulator ◽  
Braf V600e ◽  
Receptor Complex ◽  
The Core ◽  
Development Of Resistance ◽  
Biological Responses ◽  
Worse Prognosis

AbstractRNF43 is a E3 ubiquitin ligase and known negative regulator of WNT/β-catenin signaling. We demonstrate that RNF43 is also regulator of noncanonical WNT5A-induced signaling in human cells. Analysis of the RNF43 interactome using BioID and immunoprecipitation showed that RNF43 can interact with the core receptor complex components dedicated to the noncanonical Wnt pathway such as ROR1, ROR2, VANGL1 and VANGL2. RNF43 triggers VANGL2 ubiquitination and proteasomal degradation and clathrin-dependent internalization of ROR1 receptor. This activity of RNF43 is physiologically relevant and blocks pro-metastatic WNT5A signaling in melanoma. RNF43 inhibits responses to WNT5A, which results in the suppression of invasive properties of melanoma cells. Furthermore, RNF43 prevented WNT5A-assisted development of resistance to BRAF V600E inhibitor. In line with these findings, RNF43 expression decreases during melanoma progression and RNF43-low patients have worse prognosis. We conclude that RNF43 is a newly discovered negative regulator of WNT5A-mediated biological responses that desensitizes cells to WNT5A.

scholarly journals A Context-Dependent Role for the RNF146 Ubiquitin Ligase in Wingless/Wnt Signaling in Drosophila

Genetics ◽  
2018 ◽  
Vol 211 (3) ◽  
pp. 913-923 ◽  
Author(s):  
Zhenghan Wang ◽  
Ofelia Tacchelly-Benites ◽  
Geoffrey P. Noble ◽  
Megan K. Johnson ◽  
Jean-Philippe Gagné ◽  
...  
Keyword(s):  
Wnt Signaling ◽  
Ubiquitin Ligase ◽  
Wnt Pathway ◽  
Gene Activation ◽  
Negative Regulator ◽  
Compensatory Mechanisms ◽  
E3 Ligases ◽  
Pathway Activation ◽  
Wingless Signaling

Aberrant activation of the Wnt signal transduction pathway triggers the development of colorectal cancer. The ADP-ribose polymerase Tankyrase (TNKS) mediates proteolysis of Axin—a negative regulator of Wnt signaling—and provides a promising therapeutic target for Wnt-driven diseases. Proteolysis of TNKS substrates is mediated through their ubiquitination by the poly-ADP-ribose (pADPr)-dependent RING-domain E3 ubiquitin ligase RNF146/Iduna. Like TNKS, RNF146 promotes Axin proteolysis and Wnt pathway activation in some cultured cell lines, but in contrast with TNKS, RNF146 is dispensable for Axin degradation in colorectal carcinoma cells. Thus, the contexts in which RNF146 is essential for TNKS-mediated Axin destabilization and Wnt signaling remain uncertain. Herein, we tested the requirement for RNF146 in TNKS-mediated Axin proteolysis and Wnt pathway activation in a range of in vivo settings. Using null mutants in Drosophila, we provide genetic and biochemical evidence that Rnf146 and Tnks function in the same proteolysis pathway in vivo. Furthermore, like Tnks, Drosophila Rnf146 promotes Wingless signaling in multiple developmental contexts by buffering Axin levels to ensure they remain below the threshold at which Wingless signaling is inhibited. However, in contrast with Tnks, Rnf146 is dispensable for Wingless target gene activation and the Wingless-dependent control of intestinal stem cell proliferation in the adult midgut during homeostasis. Together, these findings demonstrate that the requirement for Rnf146 in Tnks-mediated Axin proteolysis and Wingless pathway activation is dependent on physiological context, and suggest that, in some cell types, functionally redundant pADPr-dependent E3 ligases or other compensatory mechanisms promote the Tnks-dependent proteolysis of Axin in both mammalian and Drosophila cells.

scholarly journals Wnt/β-Catenin/Tcf Signaling Induces the Transcription of Axin2, a Negative Regulator of the Signaling Pathway

2002 ◽  
Vol 22 (4) ◽  
pp. 1172-1183 ◽  
Author(s):  
Eek-hoon Jho ◽  
Tong Zhang ◽  
Claire Domon ◽  
Choun-Ki Joo ◽  
Jean-Noel Freund ◽  
...  
Keyword(s):  
Wnt Signaling ◽  
Signaling Pathway ◽  
Wnt Pathway ◽  
Genomic Sequence ◽  
Wnt Signaling Pathway ◽  
Genomic Region ◽  
Negative Regulator ◽  
Mobility Shift ◽  
Specific Expression

ABSTRACT Axin2/Conductin/Axil and its ortholog Axin are negative regulators of the Wnt signaling pathway, which promote the phosphorylation and degradation of β-catenin. While Axin is expressed ubiquitously, Axin2 mRNA was seen in a restricted pattern during mouse embryogenesis and organogenesis. Because many sites of Axin2 expression overlapped with those of several Wnt genes, we tested whether Axin2 was induced by Wnt signaling. Endogenous Axin2 mRNA and protein expression could be rapidly induced by activation of the Wnt pathway, and Axin2 reporter constructs, containing a 5.6-kb DNA fragment including the promoter and first intron, were also induced. This genomic region contains eight Tcf/LEF consensus binding sites, five of which are located within longer, highly conserved noncoding sequences. The mutation or deletion of these Tcf/LEF sites greatly diminished induction by β-catenin, and mutation of the Tcf/LEF site T2 abolished protein binding in an electrophoretic mobility shift assay. These results strongly suggest that Axin2 is a direct target of the Wnt pathway, mediated through Tcf/LEF factors. The 5.6-kb genomic sequence was sufficient to direct the tissue-specific expression of d2EGFP in transgenic embryos, consistent with a role for the Tcf/LEF sites and surrounding conserved sequences in the in vivo expression pattern of Axin2. Our results suggest that Axin2 participates in a negative feedback loop, which could serve to limit the duration or intensity of a Wnt-initiated signal.

scholarly journals E6AP Ubiquitin Ligase Mediates Ubiquitylation and Degradation of Hepatitis C Virus Core Protein

2006 ◽  
Vol 81 (3) ◽  
pp. 1174-1185 ◽  
Author(s):  
Masayuki Shirakura ◽  
Kyoko Murakami ◽  
Tohru Ichimura ◽  
Ryosuke Suzuki ◽  
Tetsu Shimoji ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Core Protein ◽  
Protein Levels ◽  
The Core ◽  
Hcv Core Protein ◽  
Ubiquitin Proteasome ◽  
Proteasome Pathway ◽  
Viral Nucleocapsid

ABSTRACT Hepatitis C virus (HCV) core protein is a major component of viral nucleocapsid and a multifunctional protein involved in viral pathogenesis and hepatocarcinogenesis. We previously showed that the HCV core protein is degraded through the ubiquitin-proteasome pathway. However, the molecular machinery for core ubiquitylation is unknown. Using tandem affinity purification, we identified the ubiquitin ligase E6AP as an HCV core-binding protein. E6AP was found to bind to the core protein in vitro and in vivo and promote its degradation in hepatic and nonhepatic cells. Knockdown of endogenous E6AP by RNA interference increased the HCV core protein level. In vitro and in vivo ubiquitylation assays showed that E6AP promotes ubiquitylation of the core protein. Exogenous expression of E6AP decreased intracellular core protein levels and supernatant HCV infectivity titers in the HCV JFH1-infected Huh-7 cells. Furthermore, knockdown of endogenous E6AP by RNA interference increased intracellular core protein levels and supernatant HCV infectivity titers in the HCV JFH1-infected cells. Taken together, our results provide evidence that E6AP mediates ubiquitylation and degradation of HCV core protein. We propose that the E6AP-mediated ubiquitin-proteasome pathway may affect the production of HCV particles through controlling the amounts of viral nucleocapsid protein.

scholarly journals Autophosphorylation-Induced Degradation of the Pho85 Cyclin Pcl5 Is Essential for Response to Amino Acid Limitation

2008 ◽  
Vol 28 (22) ◽  
pp. 6858-6869 ◽  
Author(s):  
Sharon Aviram ◽  
Einav Simon ◽  
Tsvia Gildor ◽  
Fabian Glaser ◽  
Daniel Kornitzer
Keyword(s):  
Amino Acid ◽  
Ubiquitin Ligase ◽  
Substrate Recognition ◽  
Amino Acid Starvation ◽  
Cyclin Dependent Kinase ◽  
Threonine Residue ◽  
The Core ◽  
Recognition Function ◽  
Scf Ubiquitin Ligase

ABSTRACT Pho85 cyclins (Pcls), activators of the yeast cyclin-dependent kinase (CDK) Pho85, belong together with the p35 activator of mammalian CDK5 to a distinct structural cyclin class. Different Pcls target Pho85 to distinct substrates. Pcl5 targets Pho85 specifically to Gcn4, a yeast transcription factor involved in the response to amino acid starvation, eventually causing the degradation of Gcn4. Pcl5 is itself highly unstable, an instability that was postulated to be important for regulation of Gcn4 degradation. We used hybrids between different Pcls to circumscribe the substrate recognition function to the core cyclin box domain of Pcl5. Furthermore, the cyclin hybrids revealed that Pcl5 degradation is uniquely dependent on two distinct degradation signals: one N-terminal and one C-terminal to the cyclin box domain. Whereas the C-terminal degradation signal is independent of Pho85, the N-terminal degradation signal requires phosphorylation of a specific threonine residue by the Pho85 molecule bound to the cyclin. This latter mode of degradation depends on the SCF ubiquitin ligase. Degradation of Pcl5 after self-catalyzed phosphorylation ensures that activity of the Pho85/Pcl5 complex is self-limiting in vivo. We demonstrate the importance of this mechanism for the regulation of Gcn4 degradation and for cell growth under conditions of amino acid starvation.

scholarly journals DET1-mediated COP1 regulation avoids HY5 activity over second-site targets to tune plant photomorphogenesis

2020 ◽  
Author(s):  
Esther Cañibano ◽  
Clara Bourbousse ◽  
Marta Garcia-Leon ◽  
Lea Wolff ◽  
Camila Garcia-Baudino ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Gene Regulatory Networks ◽  
Regulatory Networks ◽  
Target Genes ◽  
Negative Regulator ◽  
Protein Abundance ◽  
Mass Spectrometry Identification ◽  
Associated Proteins ◽  
Gene Regulatory

AbstractDE-ETIOLATED1 (DET1) is a negative regulator of plant photomorphogenesis acting as a component of the C3D complex, which can further associate to CULLIN4 to form a CRL4C3D E3 ubiquitin ligase. CRL4C3D is thought to act together with CRL4COP1SPA ubiquitin ligase, to promote the ubiquitin-mediated degradation of the master regulatory transcription factor ELONGATED HYPOCOTYL5 (HY5), thereby controlling photomorphogenic gene regulatory networks. Yet, functional links between COP1 and DET1 have long remained elusive. Here, upon mass spectrometry identification of DET1 and COP1-associated proteins, we provide in vivo evidence that DET1 associates with COP1 to promote its destabilization, a process necessary to dampen HY5 protein abundance. By regulating HY5 over-accumulation, DET1 is critical to avoid its association to second-site loci, including many PIF3 target genes. Accordingly, excessive HY5 levels result in an increased HY5 repressive activity and are sufficient to trigger fusca-like phenotypes otherwise observed typically in COP1 and COP9 signalosome mutant seedlings. This study therefore identifies that DET1-mediated regulation of COP1 stability tunes down HY5 cistrome and avoids hyper-photomorphogenic responses that might compromise plant viability.

scholarly journals The E3 Ubiquitin Ligase NARF Promotes Colony Formation in vitro and Exhibits Enhanced Expression Levels in Glioblastoma Multiforme in vivo

2010 ◽  
Vol 9 (2 and 3) ◽  
Author(s):  
Tucker Anderson ◽  
Christopher Wright ◽  
William Brooks
Keyword(s):  
Glioblastoma Multiforme ◽  
Ubiquitin Ligase ◽  
Colony Formation ◽  
Ring Finger ◽  
Negative Regulator ◽  
Low Grade ◽  
Dependent Manner ◽  
Enhanced Expression

The ubiquitin ligase NARF is a newly identified protein belonging to a small family of structurally similar E3 proteins. NARF is a negative regulator of the canonical Wnt-β-catenin pathway, targeting TCF/LEF family members for proteolytic degradation through poly-ubiquitination. We examined the role that NARF plays in cell division and found that overexpression of NARF in a colony forming assay increases colony formation in a RING finger-dependent manner. Furthermore, we demonstrate that NARF transcripts are expressed at a higher level in the grade IV brain tumor glioblastoma multiforme as compared with low grade astrocytomas. Our data thus indicate that NARF is a positive regulator of cell growth and may be involved in the tumorigenic process.

scholarly journals Usp16 modulates Wnt signaling in primary tissues through Cdkn2a regulation

2018 ◽  
Author(s):  
Maddalena Adorno ◽  
Benedetta Nicolis di Robilant ◽  
Shaheen Sikandar ◽  
Veronica Haro Acosta ◽  
Jane Antony ◽  
...  
Keyword(s):  
Wnt Signaling ◽  
Wnt Pathway ◽  
Mammary Epithelial Cells ◽  
Human Fibroblasts ◽  
Mammary Epithelial ◽  
Negative Regulator ◽  
Epithelial Regeneration ◽  
Mammary Epithelia

ABSTRACTRegulation of the Wnt pathway in stem cells and primary tissues is still poorly understood. Here we report that Usp16, a negative regulator of Bmi1/PRC1 function, modulates the Wnt pathway in mammary epithelia, primary human fibroblasts and MEFs, affecting their expansion and self-renewal potential. In mammary glands, reduced levels of Usp16 increase tissue responsiveness to Wnt, resulting in upregulation of the downstream Wnt target Axin2, expansion of the basal compartment and increased in vitro and in vivo epithelial regeneration. Usp16 regulation of the Wnt pathway in mouse and human tissues is at least in part mediated by activation of Cdkn2a, a regulator of senescence. At the molecular level, Usp16 affects Rspo-mediated phosphorylation of LRP6. In Down’s Syndrome (DS), triplication of Usp16 dampens the activation of the Wnt pathway. Usp16 copy number normalization restores normal Wnt activation in Ts65Dn mice models. Genetic upregulation of the Wnt pathway in Ts65Dn mice rescues the proliferation defect observed in mammary epithelial cells. All together, these findings link important stem cell regulators like Bmi1/Usp16 and Cdkn2a to Wnt signaling, and have implications for designing therapies for conditions, like DS, aging or degenerative diseases, where the Wnt pathway is hampered.

scholarly journals The E3 ubiquitin ligase component, Cereblon, is an evolutionarily conserved regulator of Wnt signaling

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Chen Shen ◽  
Anmada Nayak ◽  
Leif R. Neitzel ◽  
Amber A. Adams ◽  
Maya Silver-Isenstadt ◽  
...  
Keyword(s):  
Wnt Signaling ◽  
Ubiquitin Ligase ◽  
Wnt Pathway ◽  
E3 Ubiquitin Ligase ◽  
Signal Transduction Pathway ◽  
Negative Regulator ◽  
Transduction Pathway ◽  
Physiological Regulation ◽  
Cancer Cell Death ◽  
Evolutionarily Conserved

AbstractImmunomodulatory drugs (IMiDs) are important for the treatment of multiple myeloma and myelodysplastic syndrome. Binding of IMiDs to Cereblon (CRBN), the substrate receptor of the CRL4CRBN E3 ubiquitin ligase, induces cancer cell death by targeting key neo-substrates for degradation. Despite this clinical significance, the physiological regulation of CRBN remains largely unknown. Herein we demonstrate that Wnt, the extracellular ligand of an essential signal transduction pathway, promotes the CRBN-dependent degradation of a subset of proteins. These substrates include Casein kinase 1α (CK1α), a negative regulator of Wnt signaling that functions as a key component of the β-Catenin destruction complex. Wnt stimulation induces the interaction of CRBN with CK1α and its resultant ubiquitination, and in contrast with previous reports does so in the absence of an IMiD. Mechanistically, the destruction complex is critical in maintaining CK1α stability in the absence of Wnt, and in recruiting CRBN to target CK1α for degradation in response to Wnt. CRBN is required for physiological Wnt signaling, as modulation of CRBN in zebrafish and Drosophila yields Wnt-driven phenotypes. These studies demonstrate an IMiD-independent, Wnt-driven mechanism of CRBN regulation and provide a means of controlling Wnt pathway activity by CRBN, with relevance for development and disease.

ERbeta is a negative regulator of the PPARgamma in vivo

2007 ◽  
Vol 2 (S 1) ◽  
Author(s):  
A Foryst-Ludwig ◽  
M Clemenz ◽  
S Hohmann ◽  
C Sprang ◽  
N Frost ◽  
...  
Keyword(s):  
Negative Regulator
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