scholarly journals PI4K2β constrains non-canonical Wnt-PCP signalling and is localised by PAR-1 (MARK2/3) phosphorylation

2019 ◽  
Author(s):  
S. M. Tsang ◽  
William Cheng ◽  
Jingjing Li ◽  
Jeremy B. A. Green
Keyword(s):  
Planar Cell Polarity ◽  
Phosphorylation Site ◽  
Phosphatidyl Inositol ◽  
Embryonic Cell ◽  
Wnt Signalling ◽  
Reporter Assays ◽  
Canonical Wnt ◽  
Type Specification

ABSTRACTCanonical Wnt signalling is critically important in embryonic cell-type specification and cancer, while non-canonical Wnt signalling is primarily implicated in physical morphogenesis, especially planar cell polarity (PCP). Both are modulated by the polarity kinase PAR-1 (MARK2/3). PAR-1 phosphorylates the Wnt transducer Dishevelled, but there is evidence that it exerts control through other targets. Here we describe an in vitro screen for new targets of PAR-1 in which we identified phosphatidyl-inositol-4-kinase-2-beta (PI4K2β) as a substrate. Perturbation phenotypes and reporter assays in vivo show that PI4K2β inhibits both canonical and non-canonical Wnt pathways, in contrast to PI4K2α, which promotes canonical but does not affect non-canonical signalling. We show that PI4K2β acts in Wnt-responding tissue, not in Wnt production or secretion. Subcellularly, PI4K2β is cortically enriched, unlike PI4K2α, and is basolateral in polarised cells. Mutation of the PAR-1 phosphorylation site of PI4K2β mis-localises it and the endogenous core PCP protein, Vangl2. Our results reveal that PAR-1 interacts with the vertebrate PCP signalling pathway via PI4K2β.

Genetic analysis of rare coding mutations of CELSR1–3 in congenital heart and neural tube defects in Chinese people

2016 ◽  
Vol 130 (24) ◽  
pp. 2329-2340 ◽  
Author(s):  
Xiaojin Qiao ◽  
Yahui Liu ◽  
Peiqiang Li ◽  
Zhongzhong Chen ◽  
Huili Li ◽  
...  
Keyword(s):  
Neural Tube ◽  
Neural Tube Defects ◽  
Congenital Heart ◽  
Planar Cell Polarity ◽  
Heart Defects ◽  
In Vitro Assays ◽  
Organ Systems ◽  
Canonical Wnt

The planar cell polarity (PCP) pathway is critical for proper embryonic development of the neural tube and heart. Mutations in these genes have previously been implicated in the pathogenesis of neural tube defects (NTDs), but not in congenital heart defects (CHDs) in humans. We systematically identified the mutation patterns of CELSR1–3, one family of the core PCP genes, in human cohorts composed of 352 individuals with NTDs, 412 with CHDs and matched controls. A total of 72 disease-specific, rare, novel, coding mutations were identified, of which 37 were identified in patients with CHDs and 36 in patients with NTDs. Most of these mutations differed between the two cohorts, because only one novel missense mutation in CELSR1 (c.2609G>A p.P870L) was identified in both NTD and CHD patients. Both in vivo and in vitro assays revealed that CELSR1 P870L is a gain-of-function mutation. It up-regulates not only the PCP pathway, but also canonical WNT signalling in cells, and also induces both NTDs and CHDs in zebrafish embryos. As almost equal numbers of mutations were identified in each cohort, our results provided the first evidence that mutations in CELSR genes are as likely to be associated with CHDs as with NTDs, although the specific mutations differ between the two cohorts. Such differences in mutation panels suggested that CELSRs [cadherin, EGF (epidermal growth factor), LAG (laminin A G-type repeat), seven-pass receptors)] might be regulated differently during the development of these two organ systems.

scholarly journals Evolutionary Turnover in Wnt Gene Expression but Conservation of Wnt Signaling during Ovary Determination in a TSD Reptile

2021 ◽  
pp. 1-22
Author(s):  
Turk Rhen ◽  
Zachary Even ◽  
Alaina Brenner ◽  
Alexandra Lodewyk ◽  
Debojyoti Das ◽  
...  
Keyword(s):  
Sex Determination ◽  
Wnt Signaling ◽  
Planar Cell Polarity ◽  
Target Genes ◽  
Calcium Release ◽  
Canonical Wnt Signaling ◽  
Snapping Turtle ◽  
Canonical Wnt

Temperature-dependent sex determination (TSD) is a well-known characteristic of many reptilian species. However, the molecular processes linking ambient temperature to determination of gonad fate remain hazy. Here, we test the hypothesis that Wnt expression and signaling differ between female- and male-producing temperatures in the snapping turtle <i>Chelydra serpentina</i>. Canonical Wnt signaling involves secretion of glycoproteins called WNTs, which bind to and activate membrane bound receptors that trigger β-catenin stabilization and translocation to the nucleus where β-catenin interacts with TCF/LEF transcription factors to regulate expression of Wnt targets. Non-canonical Wnt signaling occurs via 2 pathways that are independent of β-catenin: one involves intracellular calcium release (the Wnt/Ca<sup>2+</sup> pathway), while the other involves activation of RAC1, JNK, and RHOA (the Wnt/planar cell polarity pathway). We screened 20 Wnt genes for differential expression between female- and male-producing temperatures during sex determination in the snapping turtle. Exposure of embryos to the female-producing temperature decreased expression of 7 Wnt genes but increased expression of 2 Wnt genes and <i>Rspo1</i> relative to embryos at the male-producing temperature. Temperature also regulated expression of putative Wnt target genes in vivo and a canonical Wnt reporter (6x TCF/LEF sites drive H2B-GFP expression) in embryonic gonadal cells in vitro. Results indicate that Wnt signaling was higher at the female- than at the male-producing temperature. Evolutionary analyses of all 20 Wnt genes revealed that thermosensitive Wnts, as opposed to insensitive Wnts, were less likely to show evidence of positive selection and experienced stronger purifying selection within TSD species.

scholarly journals WNT16 antagonises excessive canonical WNT activation and protects cartilage in osteoarthritis

2016 ◽  
Vol 76 (1) ◽  
pp. 218-226 ◽  
Author(s):  
Giovanna Nalesso ◽  
Bethan Lynne Thomas ◽  
Joanna Claire Sherwood ◽  
Jing Yu ◽  
Olga Addimanda ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Molecular Mechanisms ◽  
Wnt Signalling ◽  
Chondrocyte Apoptosis ◽  
Primary Chondrocytes ◽  
Canonical Wnt ◽  
Excessive Activation ◽  
Deficient Mice

ObjectiveBoth excessive and insufficient activation of WNT signalling results in cartilage breakdown and osteoarthritis. WNT16 is upregulated in the articular cartilage following injury and in osteoarthritis. Here, we investigate the function of WNT16 in osteoarthritis and the downstream molecular mechanisms.MethodsOsteoarthritis was induced by destabilisation of the medial meniscus in wild-type and WNT16-deficient mice. Molecular mechanisms and downstream effects were studied in vitro and in vivo in primary cartilage progenitor cells and primary chondrocytes. The pathway downstream of WNT16 was studied in primary chondrocytes and using the axis duplication assay in Xenopus.ResultsWNT16-deficient mice developed more severe osteoarthritis with reduced expression of lubricin and increased chondrocyte apoptosis. WNT16 supported the phenotype of cartilage superficial-zone progenitor cells and lubricin expression. Increased osteoarthritis in WNT16-deficient mice was associated with excessive activation of canonical WNT signalling. In vitro, high doses of WNT16 weakly activated canonical WNT signalling, but, in co-stimulation experiments, WNT16 reduced the capacity of WNT3a to activate the canonical WNT pathway. In vivo, WNT16 rescued the WNT8-induced primary axis duplication in Xenopus embryos.ConclusionsIn osteoarthritis, WNT16 maintains a balanced canonical WNT signalling and prevents detrimental excessive activation, thereby supporting the homeostasis of progenitor cells.

scholarly journals Enlisting the Ixodes scapularis Embryonic ISE6 Cell Line to Investigate the Neuronal Basis of Tick—Pathogen Interactions

Pathogens ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 70
Author(s):  
Lourdes Mateos-Hernández ◽  
Natália Pipová ◽  
Eléonore Allain ◽  
Céline Henry ◽  
Clotilde Rouxel ◽  
...  
Keyword(s):  
Cell Line ◽  
Peripheral Nerves ◽  
Ixodes Scapularis ◽  
Embryonic Cell ◽  
Cell Subpopulation ◽  
In Vivo Experiments

Neuropeptides are small signaling molecules expressed in the tick central nervous system, i.e., the synganglion. The neuronal-like Ixodes scapularis embryonic cell line, ISE6, is an effective tool frequently used for examining tick–pathogen interactions. We detected 37 neuropeptide transcripts in the I. scapularis ISE6 cell line using in silico methods, and six of these neuropeptide genes were used for experimental validation. Among these six neuropeptide genes, the tachykinin-related peptide (TRP) of ISE6 cells varied in transcript expression depending on the infection strain of the tick-borne pathogen, Anaplasma phagocytophilum. The immunocytochemistry of TRP revealed cytoplasmic expression in a prominent ISE6 cell subpopulation. The presence of TRP was also confirmed in A. phagocytophilum-infected ISE6 cells. The in situ hybridization and immunohistochemistry of TRP of I. scapularis synganglion revealed expression in distinct neuronal cells. In addition, TRP immunoreaction was detected in axons exiting the synganglion via peripheral nerves as well as in hemal nerve-associated lateral segmental organs. The characterization of a complete Ixodes neuropeptidome in ISE6 cells may serve as an effective in vitro tool to study how tick-borne pathogens interact with synganglion components that are vital to tick physiology. Therefore, our current study is a potential stepping stone for in vivo experiments to further examine the neuronal basis of tick–pathogen interactions.

scholarly journals Phosphorylation of Serine 303 Is a Prerequisite for the Stress-Inducible SUMO Modification of Heat Shock Factor 1

2003 ◽  
Vol 23 (8) ◽  
pp. 2953-2968 ◽  
Author(s):  
Ville Hietakangas ◽  
Johanna K. Ahlskog ◽  
Annika M. Jakobsson ◽  
Maria Hellesuo ◽  
Niko M. Sahlberg ◽  
...  
Keyword(s):  
Heat Shock ◽  
Phosphorylation Site ◽  
Stress Granules ◽  
Heat Shock Factor ◽  
Transcriptional Level ◽  
Heat Shock Factor 1 ◽  
Protection Mechanism ◽  
Sumo Modification

ABSTRACT The heat shock response, which is accompanied by a rapid and robust upregulation of heat shock proteins (Hsps), is a highly conserved protection mechanism against protein-damaging stress. Hsp induction is mainly regulated at transcriptional level by stress-inducible heat shock factor 1 (HSF1). Upon activation, HSF1 trimerizes, binds to DNA, concentrates in the nuclear stress granules, and undergoes a marked multisite phosphorylation, which correlates with its transcriptional activity. In this study, we show that HSF1 is modified by SUMO-1 and SUMO-2 in a stress-inducible manner. Sumoylation is rapidly and transiently enhanced on lysine 298, located in the regulatory domain of HSF1, adjacent to several critical phosphorylation sites. Sumoylation analyses of HSF1 phosphorylation site mutants reveal that specifically the phosphorylation-deficient S303 mutant remains devoid of SUMO modification in vivo and the mutant mimicking phosphorylation of S303 promotes HSF1 sumoylation in vitro, indicating that S303 phosphorylation is required for K298 sumoylation. This finding is further supported by phosphopeptide mapping and analysis with S303/7 phosphospecific antibodies, which demonstrate that serine 303 is a target for strong heat-inducible phosphorylation, corresponding to the inducible HSF1 sumoylation. A transient phosphorylation-dependent colocalization of HSF1 and SUMO-1 in nuclear stress granules provides evidence for a strictly regulated subnuclear interplay between HSF1 and SUMO.

scholarly journals Let-7a regulates EV secretion and mitochondrial oxidative phosphorylation by targeting SNAP23 in colorectal cancer

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
You Dong Liu ◽  
Xiao Peng Zhuang ◽  
Dong Lan Cai ◽  
Can Cao ◽  
Qi Sheng Gu ◽  
...  
Keyword(s):  
Oxidative Phosphorylation ◽  
Metabolic Reprogramming ◽  
Luciferase Reporter ◽  
Mitochondrial Oxidative Phosphorylation ◽  
In Vivo Assays ◽  
Reporter Assays ◽  
Extracellular Mirna

Abstract Background MicroRNAs (miRNAs) are abundant in tumor-derived extracellular vesicles (EVs) and the functions of extracellular miRNA to recipient cells have been extensively studied with tumorigenesis. However, the role of miRNA in EV secretion from cancer cells remains unknown. Methods qPCR and bioinformatics analysis were applied for determining extracellular let-7a expression from CRC patient serum and cells. Nanosight particle tracking analysis was performed for investigating the effect of let-7a on EV secretion. Luciferase reporter assays was used for identifying targeted genes synaptosome-associated protein 23 (SNAP23). In vitro and in vivo assays were used for exploring the function of let-7a/SNAP23 axis in CRC progression. Bioenergetic assays were performed for investigating the role of let-7a/SNAP23 in cellular metabolic reprogramming. Results let-7a miRNA was elevated in serum EVs from CRC patients and was enriched in CRC cell-derived EVs. We determined that let-7a could suppress EV secretion directly targeting SNAP23. In turn, SNAP23 promotes EV secretion of let-7a to downregulate the intracellular let-7a expression. In addition, we found a novel mechanism of let-7a/SNAP23 axis by regulating mitochondrial oxidative phosphorylation (OXPHOS) through Lin28a/SDHA signaling pathway. Conclusions Let-7a plays an essential role in not only inhibiting EV secretion, but also suppressing OXPHOS through SNAP23, resulting in the suppression of CRC progression, suggesting that let-7a/SNAP23 axis could provide not only effective tumor biomarkers but also novel targets for tumor therapeutic strategies.

Radiation activates the NORAD expression to promote ESCC radioimmunotherapy resistance via EEPD1/ATR/Chk1 signaling by inhibiting the pri-miR-199 processing and exosomal transfer of miR-199a-5p

2021 ◽  
Author(s):  
Yuchen Sun ◽  
Jizhao Wang ◽  
Xuanzi Sun ◽  
Jing Li ◽  
Xu Zhao ◽  
...  
Keyword(s):  
Its Region ◽  
Luciferase Reporter ◽  
In Vivo Experiments ◽  
Cycle Arrest ◽  
Non Coding Rna ◽  
Irradiation Treatment ◽  
Recombination Repair ◽  
Reporter Assays

Abstract Background Radioresistance, a poorly understood phenomenon, results in the failure of radiotherapy and consequent local recurrence, threatening a large proportion of ESCC patients. To date, lncRNAs have been found to be involved in diverse biological processes, including radioresistance.Methods ELISA was used to evaluated the H3 modifications in radio-resistant ESCC cells. FISH and qRT-PCR were adopted to examine the expression and localization of lncRNA-NORAD, pri-miR-199a and miR-199a. Electron microscopy and Nanoparticle tracking analysis (NTA) was conducted to observe and identify exosomes. High-throughput RNA sequencing and TMT mass spectrometry were performed to identify the functional lncRNAs and proteins involved in ESCC radioresistance. A series of in vitro and in vivo experiments were performed to investigate the biological effect of NORAD. CHIP, qPCR-RIP, co-IP and dual-luciferase reporter assays were used to explore the interaction of related RNAs and proteins. Results We show here that a DNA damage activated non-coding RNA-NORAD, which is critical for ESCC radio-resistance. NORAD was highly expressed in radio-resistant ESCC cells and tissues. Irradiation treatment promotes NORAD expression via enhancing H3K4me2 enrichment on its region. NORAD knockdown cells exhibit significantly hypersensitivity to irradiation in vivo and in vitro. NORAD is required for initiating repair and restart of stalled forks, G2 cycle arrest and homologous recombination repair upon irradiation treatment. Mechanistically, NORAD inhibits miR-199a expression by competitively binding PUM1 from pri-miR-199a, inhibiting the process of pri-miR-199a. Mature miR-199a in NORAD-knockdown cells can be packaged into exosomes; miR-199a restores the radiosensitivity of radioresistant cells by targeting EEPD1, then inhibiting ATR/Chk1 signaling pathway. Simultaneously, NORAD knockdown blocks the ubiquitination of PD-L1, leads to the better response for radiation and anti-PD-1 treatment in mouse model.Conclusion This study raises the possibility that LncRNA-NORAD could be a potential treatment target for improving the efficiency of immunotherapy in combination with radiation in ESCC.

scholarly journals Potential Role of microRNA-183 as a Tumor Suppressor in Hepatocellular Carcinoma

2018 ◽  
Vol 51 (5) ◽  
pp. 2065-2072 ◽  
Author(s):  
Wei Bian ◽  
Hongfei Zhang ◽  
Miao Tang ◽  
Shaojun Zhang ◽  
Lichao Wang ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Hepg2 Cells ◽  
Molecular Mechanisms ◽  
The Cancer Genome Atlas ◽  
Migration And Invasion ◽  
Metastatic Progression ◽  
Mesenchymal Transition ◽  
Reporter Assays

Background/Aims: Disseminated tumors, known as metastases, are responsible for ninety-percent of mortality due to cancer. Epithelial to mesenchymal transition, a phenomenon required for morphological conversion of non-motile discoid shaped epithelial cells to highly motile spindle-shaped mesenchymal cells, is thought to be a pre-requisite for metastatic progression. Metastasis-associated 1 (MTA1) protein is a prime inducer of EMT and metastatic progression in all solid tumors including hepatocellular carcinoma (HCC). However, the molecular mechanisms that regulate the expression and function of MTA1 in HCC have not been elucidated. Methods: In silico prediction algorithms were used to find microRNAs (miRNAs) that may target MTA1. We examined the relationship between the expression of MTA1 and miR-183 using quantitative real time PCR. We also determined the levels of the MTA1 protein using immunohistochemistry. Reporter assays, in the presence and absence of the miR-183 mimic, were used to confirm MTA1 as a bona fide target of miR183. The effect of miR-183 on HCC pathogenesis was determined using a combination of in vitro migration and invasion assay, together with in vivo xenograft experiments. The correlation between miR-183 and MTA1 expression was also studied in samples from HCC patients, and in The Cancer Genome Atlas dataset. Results: Analysis of the sequence database revealed that MTA1 is a putative target of miR-183. MTA1 protein and RNA expression showed opposite trends to miR-183 expression in breast, renal, prostate, and testicular tissue samples from cancer patients, and in the metastatic HCC cell line HepG2. An inverse correlation was also observed between MTA1 (high) and miR-183 (low) expression within samples from HHC patients and in the TCGA dataset. Reporter assays in HepG2 cells showed that miR-183 could inhibit translation of a reporter harboring the wild-type, but not the mutant miR-183 3’-untranslated region (UTR). In addition, miR-183 significantly inhibited in vitro migration and invasion in HepG2 cells, and in vivo hepatic metastasis. Conclusion: Our results reveal a novel post-transcriptional regulatory mechanism for MTA1 expression via miR-183, which is suppressed during HCC pathogenesis.

scholarly journals Regulation of GABAergic synapse formation and plasticity by GSK3β-dependent phosphorylation of gephyrin

2010 ◽  
Vol 108 (1) ◽  
pp. 379-384 ◽  
Author(s):  
Shiva K. Tyagarajan ◽  
Himanish Ghosh ◽  
Gonzalo E. Yévenes ◽  
Irina Nikonenko ◽  
Claire Ebeling ◽  
...  
Keyword(s):  
Synaptic Plasticity ◽  
Hippocampal Neurons ◽  
Glycogen Synthase ◽  
Phosphorylation Site ◽  
Scaffolding Protein ◽  
Scaffolding Proteins ◽  
Gabaergic Synapse ◽  
Gabaergic Synapses

Postsynaptic scaffolding proteins ensure efficient neurotransmission by anchoring receptors and signaling molecules in synapse-specific subcellular domains. In turn, posttranslational modifications of scaffolding proteins contribute to synaptic plasticity by remodeling the postsynaptic apparatus. Though these mechanisms are operant in glutamatergic synapses, little is known about regulation of GABAergic synapses, which mediate inhibitory transmission in the CNS. Here, we focused on gephyrin, the main scaffolding protein of GABAergic synapses. We identify a unique phosphorylation site in gephyrin, Ser270, targeted by glycogen synthase kinase 3β (GSK3β) to modulate GABAergic transmission. Abolishing Ser270 phosphorylation increased the density of gephyrin clusters and the frequency of miniature GABAergic postsynaptic currents in cultured hippocampal neurons. Enhanced, phosphorylation-dependent gephyrin clustering was also induced in vitro and in vivo with lithium chloride. Lithium is a GSK3β inhibitor used therapeutically as mood-stabilizing drug, which underscores the relevance of this posttranslational modification for synaptic plasticity. Conversely, we show that gephyrin availability for postsynaptic clustering is limited by Ca2+-dependent gephyrin cleavage by the cysteine protease calpain-1. Together, these findings identify gephyrin as synaptogenic molecule regulating GABAergic synaptic plasticity, likely contributing to the therapeutic action of lithium.

Phosphoenolpyruvate carboxylase during grape berry development: protein level, enzyme activity and regulation

2000 ◽  
Vol 27 (3) ◽  
pp. 221 ◽  
Author(s):  
Paraskevi Diakou ◽  
Laurence Svanella ◽  
Philippe Raymond ◽  
Jean-Pierre Gaudillère ◽  
Annick Moing
Keyword(s):  
Malic Acid ◽  
Protein Level ◽  
Phosphoenolpyruvate Carboxylase ◽  
Phosphorylation Site ◽  
Acid Synthesis ◽  
Grape Berry ◽  
Vitis Vinifera L ◽  
Normal Acid

The protein level and regulation of phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31, involved in malic acid synthesis) was studied during the fruit development of two grape (Vitis vinifera L.) varieties, ‘Cabernet Sauvignon’ and ‘Gora Chirine’, with berries of normal and low organic acid content, respectively. The protein level and in vitro activity were higher in the low-acid variety than in the normal-acid variety for most stages. In vivo PEPC activity, measured using 14 CO2 labelling, was significantly higher in the low-acid variety than in the normal-acid variety about 1 week before and 1 week after veraison (the day which corresponds to the onset of ripening). However, partitioning into malate was the same for both varieties. Antibodies raised against the N-terminal part of SorghumPEPC recognised the grape berry PEPC, indicating the presence of the consensus phosphorylation site involved in PEPC regulation. PEPC phosphorylation status was estimated by studying sensitivity to pH and malate. Grape berry PEPC appeared more sensitive to low pH and malate during ripening (IC50 malate, 0.2–0.7 mM) compared to during the earlier stages of development (IC50 malate, 1.2–2 mM) for both varieties. Therefore, in the normal-acid variety, PEPC seems to participate in controlling malic acid accumulation but does not seem to control the differences in malic acid concentration observed between the two varieties.

Sign in / Sign up

Export Citation Format

Share Document