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The Journal of Cell Biology Sep 2020The tumor suppressor PTEN is essential for early development. Its lipid phosphatase activity converts PIP3 to PIP2 and antagonizes the PI3K-Akt pathway. In this study,...
The tumor suppressor PTEN is essential for early development. Its lipid phosphatase activity converts PIP3 to PIP2 and antagonizes the PI3K-Akt pathway. In this study, we demonstrate that PTEN's protein phosphatase activity is required for epiblast epithelial differentiation and polarization. This is accomplished by reconstitution of PTEN-null embryoid bodies with PTEN mutants that lack only PTEN's lipid phosphatase activity or both PTEN's lipid and protein phosphatase activities. Phosphotyrosine antibody immunoprecipitation and mass spectrometry were used to identify Abi1, a core component of the WASP-family verprolin homologous protein (WAVE) regulatory complex (WRC), as a new PTEN substrate. We demonstrate that PTEN dephosphorylation of Abi1 at Y213 and S216 results in Abi1 degradation through the calpain pathway. This leads to down-regulation of the WRC and reorganization of the actin cytoskeleton. The latter is critical to the transformation of nonpolar pluripotent stem cells into the polarized epiblast epithelium. Our findings establish a link between PTEN and WAVE-Arp2/3-regulated actin cytoskeletal dynamics in epithelial morphogenesis.
Topics: Actin Cytoskeleton; Adaptor Proteins, Signal Transducing; Animals; Calpain; Cell Differentiation; Cell Line, Tumor; Cytoskeletal Proteins; Down-Regulation; Epithelial Cells; Epithelium; Female; Germ Layers; Humans; Mice; Mice, Inbred C57BL; Morphogenesis; PTEN Phosphohydrolase; Pregnancy; Signal Transduction
PubMed: 32673396
DOI: 10.1083/jcb.201910041 -
Journal of Virology Jul 2018Hepatitis B virus (HBV) core protein consists of an N-terminal assembly domain and a C-terminal domain (CTD) with seven conserved serines or threonines that are...
Hepatitis B virus (HBV) core protein consists of an N-terminal assembly domain and a C-terminal domain (CTD) with seven conserved serines or threonines that are dynamically phosphorylated/dephosphorylated during the viral replication cycle. Sulfamoylbenzamide derivatives are small molecular core protein allosteric modulators (CpAMs) that bind to the heteroaryldihydropyrimidine (HAP) pocket between the core protein dimer-dimer interfaces. CpAM binding alters the kinetics and pathway of capsid assembly and can result in the formation of morphologically "normal" capsids devoid of viral pregenomic RNA (pgRNA) and DNA polymerase. In order to investigate the mechanism underlying CpAM inhibition of pgRNA encapsidation, we developed an immunoblotting assay that can resolve core protein based on its phosphorylation status and demonstrated, for the first time, that core protein is hyperphosphorylated in free dimers and empty capsids from both mock-treated and CpAM-treated cells but is hypophosphorylated in pgRNA- and DNA-containing nucleocapsids. Interestingly, inhibition of pgRNA encapsidation by a heat shock protein 90 (HSP90) inhibitor prevented core protein dephosphorylation. Moreover, core proteins with point mutations at the wall of the HAP pocket, V124A and V124W, assembled empty capsids and nucleocapsids with altered phosphorylation status. The results thus suggest that core protein dephosphorylation occurs in the assembly of pgRNA and that interference with the interaction between core protein subunits at dimer-dimer interfaces during nucleocapsid assembly alters not only capsid structure, but also core protein dephosphorylation. Hence, inhibition of pgRNA encapsidation by CpAMs might be due to disruption of core protein dephosphorylation during nucleocapsid assembly. Dynamic phosphorylation of HBV core protein regulates multiple steps of viral replication. However, the regulatory function was mainly investigated by phosphomimetic mutagenesis, which disrupts the natural dynamics of core protein phosphorylation/dephosphorylation. Development of an immunoblotting assay capable of resolving hyper- and hypophosphorylated core proteins allowed us to track the phosphorylation status of core proteins existing as free dimers and the variety of intracellular capsids and to investigate the role of core protein phosphorylation/dephosphorylation in viral replication. Here, we found that disruption of core protein interaction at dimer-dimer interfaces during nucleocapsid assembly (by CpAMs or mutagenesis) inhibited core protein dephosphorylation and pgRNA packaging. Our work has thus revealed a novel function of core protein dephosphorylation in HBV replication and the mechanism by which CpAMs, a class of compounds that are currently in clinical trials for treatment of chronic hepatitis B, induce the assembly of empty capsids.
Topics: Allosteric Regulation; Capsid; Cells, Cultured; Genome, Viral; Hepatitis B; Hepatitis B virus; Hepatocytes; Humans; Phosphorylation; RNA Precursors; RNA, Viral; Viral Core Proteins; Virus Assembly; Virus Replication
PubMed: 29669831
DOI: 10.1128/JVI.02139-17 -
Theranostics 2024: Meningeal lymphatic vessels (MLVs) are essential for the clearance of subdural hematoma (SDH). However, SDH impairs their drainage function, and the pathogenesis...
: Meningeal lymphatic vessels (MLVs) are essential for the clearance of subdural hematoma (SDH). However, SDH impairs their drainage function, and the pathogenesis remains unclear. Herein, we aimed to understand the pathological mechanisms of MLV dysfunction following SDH and to test whether atorvastatin, an effective drug for SDH clearance, improves meningeal lymphatic drainage (MLD). : We induced SDH models in rats by injecting autologous blood into the subdural space and evaluated MLD using Gadopentetate D, Evans blue, and CFSE-labeled erythrocytes. Whole-mount immunofluorescence and transmission electron microscopy were utilized to detect the morphology of MLVs. Phosphoproteomics, western blot, flow cytometry, and experiments were performed to investigate the molecular mechanisms underlying dysfunctional MLVs. : The basal MLVs were detected to have abundant valves and play an important role in draining subdural substances. Following SDH, these basal MLVs exhibited disrupted endothelial junctions and dilated lumen, leading to impaired MLD. Subsequent proteomics analysis of the meninges detected numerous dephosphorylated proteins, primarily enriched in the adherens junction, including significant dephosphorylation of ERK1/2 within the meningeal lymphatic endothelial cells (LECs). Subdural injection of the ERK1/2 kinase inhibitor PD98059 resulted in dilated basal MLVs and impaired MLD, resembling the dysfunctional MLVs observed in SDH. Moreover, inhibiting ERK1/2 signaling severely disrupted intercellular junctions between cultured LECs. Finally, atorvastatin was revealed to protect the structure of basal MLVs and accelerate MLD following SDH. However, these beneficial effects of atorvastatin were abolished when combined with PD98059. : Our findings demonstrate that SDH induces ERK1/2 dephosphorylation in meningeal LECs, leading to disrupted basal MLVs and impaired MLD. Additionally, we reveal a beneficial effect of atorvastatin in improving MLD.
Topics: Rats; Animals; Glymphatic System; Atorvastatin; Endothelial Cells; MAP Kinase Signaling System; Hematoma, Subdural; Lymphatic Vessels
PubMed: 38164141
DOI: 10.7150/thno.87633 -
Molecular Biology of the Cell Mar 2022Transcriptional factor EB (TFEB) is a master regulator of genes required for autophagy and lysosomal function. The nuclear localization of TFEB is blocked by the...
Transcriptional factor EB (TFEB) is a master regulator of genes required for autophagy and lysosomal function. The nuclear localization of TFEB is blocked by the mechanistic target of rapamycin complex 1 (mTORC1)-dependent phosphorylation of TFEB at multiple sites including Ser-211. Here we show that inhibition of PIKfyve, which produces phosphatidylinositol 3,5-bisphosphate on endosomes and lysosomes, causes a loss of Ser-211 phosphorylation and concomitant nuclear localization of TFEB. We found that while mTORC1 activity toward S6K1, as well as other major mTORC1 substrates, is not impaired, PIKfyve inhibition specifically impedes the interaction of TFEB with mTORC1. This suggests that mTORC1 activity on TFEB is selectively inhibited due to loss of mTORC1 access to TFEB. In addition, we found that TFEB activation during inhibition of PIKfyve relies on the ability of protein phosphatase 2A (PP2A) but not calcineurin/PPP3 to dephosphorylate TFEB Ser-211. Thus when PIKfyve is inhibited, PP2A is dominant over mTORC1 for control of TFEB phosphorylation at Ser-S211. Together these findings suggest that mTORC1 and PP2A have opposing roles on TFEB via phosphorylation and dephosphorylation of Ser-211, respectively, and further that PIKfyve inhibits TFEB activity by facilitating mTORC1-dependent phosphorylation of TFEB.
Topics: Autophagy; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; Lysosomes; Mechanistic Target of Rapamycin Complex 1; Phosphorylation; Protein Phosphatase 2
PubMed: 35020443
DOI: 10.1091/mbc.E21-06-0309 -
Frontiers in Plant Science 2022Glycogen synthase kinases, also known as SHAGGY-like Kinases (GSKs/SKs), are highly conserved serine/threonine protein kinases present both in animals and plants. Plant... (Review)
Review
Glycogen synthase kinases in model and crop plants - From negative regulators of brassinosteroid signaling to multifaceted hubs of various signaling pathways and modulators of plant reproduction and yield.
Glycogen synthase kinases, also known as SHAGGY-like Kinases (GSKs/SKs), are highly conserved serine/threonine protein kinases present both in animals and plants. Plant genomes contain multiple homologs of the genes which participate in various biological processes. Plant GSKs/SKs, and their best known representative in Brassinosteroid Insentisive2 (BIN2/SK21) in particular, were first identified as components of the brassinosteroid (BR) signaling pathway. As phytohormones, BRs regulate a wide range of physiological processes in plants - from germination, cell division, elongation and differentiation to leaf senescence, and response to environmental stresses. The GSKs/SKs proteins belong to a group of several highly conserved components of the BR signaling which evolved early during evolution of this molecular relay. However, recent reports indicated that the GSKs/SKs proteins are also implicated in signaling pathways of other phytohormones and stress-response processes. As a consequence, the GSKs/SKs proteins became hubs of various signaling pathways and modulators of plant development and reproduction. Thus, it is very important to understand molecular mechanisms regulating activity of the GSKs/SKs proteins, but also to get insights into role of the GSKs/SKs proteins in modulation of stability and activity of various substrate proteins which participate in the numerous signaling pathways. Although elucidation of these aspects is still in progress, this review presents a comprehensive and detailed description of these processes and their implications for regulation of development, stress response, and reproduction of model and crop species. The GSKs/SKs proteins and their activity are modulated through phosphorylation and de-phosphorylation reactions which are regulated by various proteins. Importantly, both phosphorylations and de-phosphorylations may have positive and negative effects on the activity of the GSKs/SKs proteins. Additionally, the activity of the GSKs/SKs proteins is positively regulated by reactive oxygen species, whereas it is negatively regulated through ubiquitylation, deacetylation, and nitric oxide-mediated nitrosylation. On the other hand, the GSKs/SKs proteins interact with proteins representing various signaling pathways, and on the basis of the complicated network of interactions the GSKs/SKs proteins differentially regulate various physiological, developmental, stress response, and yield-related processes.
PubMed: 35909730
DOI: 10.3389/fpls.2022.939487 -
Cell & Bioscience 2019Dual-specificity phosphatases (DUSPs) are a subset of protein tyrosine phosphatases (PTPs), many of which dephosphorylate the residues of phosphor-serine/threonine and... (Review)
Review
Dual-specificity phosphatases (DUSPs) are a subset of protein tyrosine phosphatases (PTPs), many of which dephosphorylate the residues of phosphor-serine/threonine and phosphor-tyrosine on mitogen-activated protein kinases (MAPKs), and hence are also referred to as MAPK phosphatases (MKPs). Homologue of Vaccinia virus H1 phosphatase gene clone 5 (HVH-5), also known as DUSP8, is a unique member of the DUSPs family of phosphatases. Accumulating evidence has shown that DUSP8 plays an important role in phosphorylation-mediated signal transduction of MAPK signaling ranging from cell oxidative stress response, cell apoptosis and various human diseases. It is generally believed that DUSP8 exhibits significant dephosphorylation activity against JNK, however, with the deepening of research, plenty of new literature reports that DUSP8 also has effective dephosphorylation activity on p38 MAPK and ERKs, successfully affects the transduction of MAPKs pathway, indicating that DUSP8 presents a unknown diversity of DUSPs family on distinct corresponding dephosphorylated substrates in different biological events. Therefore, the in-depth study of DUSP8 not only throws a new light on the multi-biological function of DUSPs, but also is much valuable for the reveal of complex pathobiology of clinical diseases. In this review, we provide a detail overview of DUSP8 phosphatase structure, biological function and expression regulation, as well as its role in related clinical human diseases, which might be help for the understanding of biological function of DUSP8 and the development of prevention, diagnosis and therapeutics in related human diseases.
PubMed: 31467668
DOI: 10.1186/s13578-019-0329-4 -
Frontiers in Plant Science 2022Serine/threonine protein phosphatase 2C (PP2C) dephosphorylates proteins and plays crucial roles in plant growth, development, and stress response. In this study, we...
Serine/threonine protein phosphatase 2C (PP2C) dephosphorylates proteins and plays crucial roles in plant growth, development, and stress response. In this study, we characterized a clade B member of maize PP2C family, i.e., ZmPP2C26, that negatively regulated drought tolerance by dephosphorylating ZmMAPK3 and ZmMAPK7 in maize. The gene generated and isoforms through untypical alternative splicing. ZmPP2C26S lost 71 amino acids including an MAPK interaction motif and showed higher phosphatase activity than ZmPP2C26L. ZmPP2C26L directly interacted with, dephosphorylated ZmMAPK3 and ZmMAPK7, and localized in chloroplast and nucleus, but ZmPP2C26S only dephosphorylated ZmMAPK3 and localized in cytosol and nucleus. The expression of and was significantly inhibited by drought stress. Meanwhile, the maize mutant exhibited enhancement of drought tolerance with higher root length, root weight, chlorophyll content, and photosynthetic rate compared with wild type. However, overexpression of and significantly decreased drought tolerance in and rice with lower root length, chlorophyll content, and photosynthetic rate. Phosphoproteomic analysis revealed that the ZmPP2C26 protein also altered phosphorylation level of proteins involved in photosynthesis. This study provides insights into understanding the mechanism of PP2C in response to abiotic stress.
PubMed: 35463404
DOI: 10.3389/fpls.2022.851531 -
Cardiovascular Research Jan 2022Cardiovascular calcification (CVC) is associated with increased morbidity and mortality. It develops in several diseases and locations, such as in the tunica intima in... (Review)
Review
Cardiovascular calcification (CVC) is associated with increased morbidity and mortality. It develops in several diseases and locations, such as in the tunica intima in atherosclerosis plaques, in the tunica media in type 2 diabetes and chronic kidney disease, and in aortic valves. In spite of the wide occurrence of CVC and its detrimental effects on cardiovascular diseases (CVD), no treatment is yet available. Most of CVC involve mechanisms similar to those occurring during endochondral and/or intramembranous ossification. Logically, since tissue-nonspecific alkaline phosphatase (TNAP) is the key-enzyme responsible for skeletal/dental mineralization, it is a promising target to limit CVC. Tools have recently been developed to inhibit its activity and preclinical studies conducted in animal models of vascular calcification already provided promising results. Nevertheless, as its name indicates, TNAP is ubiquitous and recent data indicate that it dephosphorylates different substrates in vivo to participate in other important physiological functions besides mineralization. For instance, TNAP is involved in the metabolism of pyridoxal phosphate and the production of neurotransmitters. TNAP has also been described as an anti-inflammatory enzyme able to dephosphorylate adenosine nucleotides and lipopolysaccharide. A better understanding of the full spectrum of TNAP's functions is needed to better characterize the effects of TNAP inhibition in diseases associated with CVC. In this review, after a brief description of the different types of CVC, we describe the newly uncovered additional functions of TNAP and discuss the expected consequences of its systemic inhibition in vivo.
Topics: Alkaline Phosphatase; Animals; Arteries; Cardiovascular Agents; Enzyme Inhibitors; Humans; Phosphorylation; Signal Transduction; Substrate Specificity; Vascular Calcification
PubMed: 33070177
DOI: 10.1093/cvr/cvaa299 -
Turkish Neurosurgery 2024To elucidate the function of ASK-1 and the role of its modulators in the induction of Temozolamide (TMZ) resistance in glioma and the underlying mechanism.
AIM
To elucidate the function of ASK-1 and the role of its modulators in the induction of Temozolamide (TMZ) resistance in glioma and the underlying mechanism.
MATERIAL AND METHODS
ASK-1 phosphorylation, the IC50 of TMZ, cell viability, and apoptosis were assessed in the U87 and U251 glioma cell lines and the derived TMZ-resistant cell lines U87-TR and U251-TR. We then blocked ASK-1 function, either with an inhibitor or by overexpression of multiple ASK-1 upstream modulators, to further explore the role of ASK-1 in TMZ-resistant glioma.
RESULTS
TMZ-resistant glioma cells showed high IC50 values of TMZ, high survival, and low levels of apoptosis following the TMZ challenge. ASK-1 phosphorylation, but not protein expression, was higher in U87 and U251 cells than in TMZ-resistant glioma cells exposed to TMZ. The addition of the ASK-1 inhibitor selonsertib (SEL) resulted in the dephosphorylation of ASK-1 in U87 and U251 cells after the TMZ challenge. SEL treatment increased the TMZ resistance of U87 and U251 cells, as evidenced by the increased IC50 and cell survival rate and low apoptosis rate. Overexpression of some ASK-1 upstream suppressors [Thioredoxin (Trx), protein phosphatase 5 (PP5), 14-3-3, and cell division cycle 25C (Cdc25C)] led to various degrees of ASK-1 dephosphorylation and a TMZresistant phenotype in U87 and U251 cells.
CONCLUSION
Dephosphorylation of ASK-1 induced TMZ resistance in human glioma cells, and several ASK-1 upstream suppressors, including Trx, PP5, 14-3-3, and Cdc25C, are involved in this phenotypic change induced by dephosphorylation of ASK-1.
Topics: Humans; Antineoplastic Agents, Alkylating; Apoptosis; Brain Neoplasms; Cell Line, Tumor; Drug Resistance, Neoplasm; Glioma; Temozolomide
PubMed: 36951032
DOI: 10.5137/1019-5149.JTN.41212-22.1 -
Molecular Cell Oct 2022Neuronal activity induces topoisomerase IIβ (Top2B) to generate DNA double-strand breaks (DSBs) within the promoters of neuronal early response genes (ERGs) and...
Neuronal activity induces topoisomerase IIβ (Top2B) to generate DNA double-strand breaks (DSBs) within the promoters of neuronal early response genes (ERGs) and facilitate their transcription, and yet, the mechanisms that control Top2B-mediated DSB formation are unknown. Here, we report that stimulus-dependent calcium influx through NMDA receptors activates the phosphatase calcineurin to dephosphorylate Top2B at residues S1509 and S1511, which stimulates its DNA cleavage activity and induces it to form DSBs. Exposing mice to a fear conditioning paradigm also triggers Top2B dephosphorylation at S1509 and S1511 in the hippocampus, indicating that calcineurin also regulates Top2B-mediated DSB formation following physiological neuronal activity. Furthermore, calcineurin-Top2B interactions following neuronal activity and sites that incur activity-induced DSBs are preferentially localized at the nuclear periphery in neurons. Together, these results reveal how radial gene positioning and the compartmentalization of activity-dependent signaling govern the position and timing of activity-induced DSBs and regulate gene expression patterns in neurons.
Topics: Animals; Mice; Calcineurin; Calcium; DNA; DNA Breaks, Double-Stranded; DNA Topoisomerases, Type II; DNA-Binding Proteins; Neurons; Receptors, N-Methyl-D-Aspartate
PubMed: 36206766
DOI: 10.1016/j.molcel.2022.09.012