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1.
PLoS One ; 15(8): e0228002, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32764831

RESUMO

Irinotecan specifically targets topoisomerase I (topoI), and is used to treat various solid tumors, but only 13-32% of patients respond to the therapy. Now, it is understood that the rapid rate of topoI degradation in response to irinotecan causes irinotecan resistance. We have published that the deregulated DNA-PKcs kinase cascade ensures rapid degradation of topoI and is at the core of the drug resistance mechanism of topoI inhibitors, including irinotecan. We also identified CTD small phosphatase 1 (CTDSP1) (a nuclear phosphatase) as a primary upstream regulator of DNA-PKcs in response to topoI inhibitors. Previous reports showed that rabeprazole, a proton pump inhibitor (PPI) inhibits CTDSP1 activity. The purpose of this study was to confirm the effects of rabeprazole on CTDSP1 activity and its impact on irinotecan-based therapy in colon cancer. Using differentially expressing CTDSP1 cells, we demonstrated that CTDSP1 contributes to the irinotecan sensitivity by preventing topoI degradation. Retrospective analysis of patients receiving irinotecan with or without rabeprazole has shown the effects of CTDSP1 on irinotecan response. These results indicate that CTDSP1 promotes sensitivity to irinotecan and rabeprazole prevents this effect, resulting in drug resistance. To ensure the best chance at effective treatment, rabeprazole may not be a suitable PPI for cancer patients treated with irinotecan.


Assuntos
Neoplasias Colorretais/metabolismo , DNA Topoisomerases Tipo I/metabolismo , Rabeprazol/metabolismo , Linhagem Celular Tumoral , Neoplasias do Colo/metabolismo , Neoplasias Colorretais/fisiopatologia , DNA , DNA Topoisomerases Tipo I/fisiologia , Proteína Quinase Ativada por DNA/metabolismo , Resistência a Medicamentos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/fisiologia , Humanos , Irinotecano/metabolismo , Irinotecano/farmacologia , Fosfoproteínas Fosfatases/antagonistas & inibidores , Fosfoproteínas Fosfatases/metabolismo , Inibidores da Bomba de Prótons/farmacologia , Rabeprazol/farmacologia , Estudos Retrospectivos , Inibidores da Topoisomerase I/farmacologia
2.
Proc Natl Acad Sci U S A ; 117(30): 17796-17807, 2020 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-32651268

RESUMO

Fluctuation in signal transduction pathways is frequently observed during mammalian development. However, its role in regulating stem cells has not been explored. Here we tracked spatiotemporal ERK MAPK dynamics in human epidermal stem cells. While stem cells and differentiated cells were distinguished by high and low stable basal ERK activity, respectively, we also found cells with pulsatile ERK activity. Transitions from Basalhi-Pulselo (stem) to Basalhi-Pulsehi, Basalmid-Pulsehi, and Basallo-Pulselo (differentiated) cells occurred in expanding keratinocyte colonies and in response to differentiation stimuli. Pharmacological inhibition of ERK induced differentiation only when cells were in the Basalmid-Pulsehi state. Basal ERK activity and pulses were differentially regulated by DUSP10 and DUSP6, leading us to speculate that DUSP6-mediated ERK pulse down-regulation promotes initiation of differentiation, whereas DUSP10-mediated down-regulation of mean ERK activity promotes and stabilizes postcommitment differentiation. Levels of MAPK1/MAPK3 transcripts correlated with DUSP6 and DUSP10 transcripts in individual cells, suggesting that ERK activity is negatively regulated by transcriptional and posttranslational mechanisms. When cells were cultured on a topography that mimics the epidermal-dermal interface, spatial segregation of mean ERK activity and pulses was observed. In vivo imaging of mouse epidermis revealed a patterned distribution of basal cells with pulsatile ERK activity, and down-regulation was linked to the onset of differentiation. Our findings demonstrate that ERK MAPK signal fluctuations link kinase activity to stem cell dynamics.


Assuntos
Diferenciação Celular , Células Epidérmicas/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células-Tronco/metabolismo , Animais , Técnicas de Cultura de Células , Proliferação de Células , Ativação Enzimática , Células Epidérmicas/citologia , Queratinócitos/metabolismo , Mamíferos , Camundongos , Fosfoproteínas Fosfatases/metabolismo , Transdução de Sinais , Células-Tronco/citologia
3.
Proc Natl Acad Sci U S A ; 117(26): 15123-15131, 2020 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-32541056

RESUMO

Yeast form complex highly organized colonies in which cells undergo spatiotemporal phenotypic differentiation in response to local gradients of nutrients, metabolites, and specific signaling molecules. Colony fitness depends on cell interactions, cooperation, and the division of labor between differentiated cell subpopulations. Here, we describe the regulation and dynamics of the expansion of papillae that arise during colony aging, which consist of cells that overcome colony regulatory rules and disrupt the synchronized colony structure. We show that papillae specifically expand within the U cell subpopulation in differentiated colonies. Papillae emerge more frequently in some strains than in others. Genomic analyses further revealed that the Whi2p-Psr1p/Psr2p complex (WPPC) plays a key role in papillae expansion. We show that cells lacking a functional WPPC have a sizable interaction-specific fitness advantage attributable to production of and resistance to a diffusible compound that inhibits growth of other cells. Competitive superiority and high relative fitness of whi2 and psr1psr2 strains are particularly pronounced in dense spatially structured colonies and are independent of TORC1 and Msn2p/Msn4p regulators previously associated with the WPPC function. The WPPC function, described here, might be a regulatory mechanism that balances cell competition and cooperation in dense yeast populations and, thus, contributes to cell synchronization, pattern formation, and the expansion of cells with a competitive fitness advantage.


Assuntos
Proliferação de Células/fisiologia , Regulação Fúngica da Expressão Gênica/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Transdução de Sinais/fisiologia
4.
Nat Commun ; 11(1): 2549, 2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32439975

RESUMO

Mitochondria undergo dynamic fusion/fission, biogenesis and mitophagy in response to stimuli or stresses. Disruption of mitochondrial homeostasis could lead to cell senescence, although the underlying mechanism remains unclear. We show that deletion of mitochondrial phosphatase PGAM5 leads to accelerated retinal pigment epithelial (RPE) senescence in vitro and in vivo. Mechanistically, PGAM5 is required for mitochondrial fission through dephosphorylating DRP1. PGAM5 deletion leads to increased mitochondrial fusion and decreased mitochondrial turnover. As results, cellular ATP and reactive oxygen species (ROS) levels are elevated, mTOR and IRF/IFN-ß signaling pathways are enhanced, leading to cellular senescence. Overexpression of Drp1 K38A or S637A mutant phenocopies or rescues mTOR activation and senescence in PGAM5-/- cells, respectively. Young but not aging Pgam5-/- mice are resistant to sodium iodate-induced RPE cell death. Our studies establish a link between defective mitochondrial fission, cellular senescence and age-dependent oxidative stress response, which have implications in age-related diseases.


Assuntos
Senescência Celular , Dinâmica Mitocondrial , Fosfoproteínas Fosfatases/metabolismo , Fatores Etários , Animais , Linhagem Celular , Dinaminas/genética , Dinaminas/metabolismo , Regulação da Expressão Gênica , Humanos , Camundongos , Camundongos Knockout , Mitocôndrias/metabolismo , Estresse Oxidativo/genética , Fosfoproteínas Fosfatases/genética , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/patologia , Transdução de Sinais
5.
PLoS Genet ; 16(4): e1008735, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32251417

RESUMO

The protein phosphatase Cdc25 is a key regulator of the cell cycle by activating Cdk-cyclin complexes. Cdc25 is regulated by its expression levels and post-translational mechanisms. In early Drosophila embryogenesis, Cdc25/Twine drives the fast and synchronous nuclear cycles. A pause in the cell cycle and the remodeling to a more generic cell cycle mode with a gap phase are determined by Twine inactivation and destruction in early interphase 14, in response to zygotic genome activation. Although the pseudokinase Tribbles contributes to the timely degradation of Twine, Twine levels are controlled by additional yet unknown post-translational mechanisms. Here, we apply a non-invasive method based on fluorescence fluctuation analysis (FFA) to record the absolute concentration profiles of Twine with minute-scale resolution in single living embryos. Employing this assay, we found that Protein phosphatase V (PpV), the homologue of the catalytic subunit of human PP6, ensures appropriately low Twine protein levels at the onset of interphase 14. PpV controls directly or indirectly the phosphorylation of Twine at multiple serine and threonine residues as revealed by phosphosite mapping. Mutational analysis confirmed that these sites are involved in control of Twine protein dynamics, and cell cycle remodeling is delayed in a fraction of the phosphosite mutant embryos. Our data reveal a novel mechanism for control of Twine protein levels and their significance for embryonic cell cycle remodeling.


Assuntos
Proteínas de Drosophila/genética , Embrião não Mamífero/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Animais , Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Drosophila , Proteínas de Drosophila/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Microscopia de Fluorescência/métodos , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Proteólise
6.
PLoS Genet ; 16(4): e1008324, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32287271

RESUMO

Transposable elements (TEs) are DNA repeats that must remain silenced to ensure cell integrity. Several epigenetic pathways including DNA methylation and histone modifications are involved in the silencing of TEs, and in the regulation of gene expression. In Arabidopsis thaliana, the TE-derived plant mobile domain (PMD) proteins have been involved in TE silencing, genome stability, and control of developmental processes. Using a forward genetic screen, we found that the PMD protein MAINTENANCE OF MERISTEMS (MAIN) acts synergistically and redundantly with DNA methylation to silence TEs. We found that MAIN and its close homolog MAIN-LIKE 1 (MAIL1) interact together, as well as with the phosphoprotein phosphatase (PPP) PP7-like (PP7L). Remarkably, main, mail1, pp7l single and mail1 pp7l double mutants display similar developmental phenotypes, and share common subsets of upregulated TEs and misregulated genes. Finally, phylogenetic analyses of PMD and PP7-type PPP domains among the Eudicot lineage suggest neo-association processes between the two protein domains to potentially generate new protein function. We propose that, through this interaction, the PMD and PPP domains may constitute a functional protein module required for the proper expression of a common set of genes, and for silencing of TEs.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Elementos de DNA Transponíveis/genética , Regulação da Expressão Gênica de Plantas , Inativação Gênica , Proteínas Nucleares/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Metilação de DNA , Epigênese Genética , Heterocromatina/metabolismo , Mutação , Proteínas Nucleares/genética , Fosfoproteínas Fosfatases/genética , Ligação Proteica , Domínios Proteicos , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo
7.
Arterioscler Thromb Vasc Biol ; 40(7): 1705-1721, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32268790

RESUMO

OBJECTIVE: A decrease in nitric oxide, leading to vascular smooth muscle cell proliferation, is a common pathological feature of vascular proliferative diseases. Nitric oxide synthesis by eNOS (endothelial nitric oxide synthase) is precisely regulated by protein kinases including AKT1. ENH (enigma homolog protein) is a scaffolding protein for multiple protein kinases, but whether it regulates eNOS activation and vascular remodeling remains unknown. Approach and Results: ENH was upregulated in injured mouse arteries and human atherosclerotic plaques and was associated with coronary artery disease. Neointima formation in carotid arteries, induced by ligation or wire injury, was greatly decreased in endothelium-specific ENH-knockout mice. Vascular ligation reduced AKT and eNOS phosphorylation and nitric oxide production in the endothelium of control but not ENH-knockout mice. ENH was found to interact with AKT1 and its phosphatase PHLPP2 (pleckstrin homology domain and leucine-rich repeat protein phosphatase 2). AKT and eNOS activation were prolonged in VEGF (vascular endothelial growth factor)-induced ENH- or PHLPP2-deficient endothelial cells. Inhibitors of either AKT or eNOS effectively restored ligation-induced neointima formation in ENH-knockout mice. Moreover, endothelium-specific PHLPP2-knockout mice displayed reduced ligation-induced neointima formation. Finally, PHLPP2 was increased in the endothelia of human atherosclerotic plaques and blood cells from patients with coronary artery disease. CONCLUSIONS: ENH forms a complex with AKT1 and its phosphatase PHLPP2 to negatively regulate AKT1 activation in the artery endothelium. AKT1 deactivation, a decrease in nitric oxide generation, and subsequent neointima formation induced by vascular injury are mediated by ENH and PHLPP2. ENH and PHLPP2 are thus new proatherosclerotic factors that could be therapeutically targeted.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Lesões das Artérias Carótidas/enzimologia , Artéria Carótida Primitiva/enzimologia , Proteínas dos Microfilamentos/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Remodelação Vascular , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Aterosclerose/enzimologia , Aterosclerose/patologia , Aterosclerose/fisiopatologia , Lesões das Artérias Carótidas/genética , Lesões das Artérias Carótidas/patologia , Lesões das Artérias Carótidas/fisiopatologia , Artéria Carótida Primitiva/patologia , Artéria Carótida Primitiva/fisiopatologia , Células Cultivadas , Doença da Artéria Coronariana/enzimologia , Doença da Artéria Coronariana/patologia , Doença da Artéria Coronariana/fisiopatologia , Modelos Animais de Doenças , Células Endoteliais da Veia Umbilical Humana/enzimologia , Humanos , Proteínas com Domínio LIM/genética , Proteínas com Domínio LIM/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas dos Microfilamentos/deficiência , Proteínas dos Microfilamentos/genética , Neointima , Óxido Nítrico/metabolismo , Fosfoproteínas Fosfatases/deficiência , Fosfoproteínas Fosfatases/genética , Fosforilação , Transdução de Sinais
8.
Biochim Biophys Acta Mol Cell Res ; 1867(8): 118727, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32339526

RESUMO

The Ser/Thr protein phosphatase Ppz1 from Saccharomyces cerevisiae is the best characterized member of a family of enzymes only found in fungi. Ppz1 is regulated in vivo by two inhibitory subunits, Hal3 and Vhs3, which are moonlighting proteins also involved in the decarboxylation of the 4-phosphopantothenoylcysteine (PPC) intermediate required for coenzyme A biosynthesis. It has been reported that, when overexpressed, Ppz1 is the most toxic protein in yeast. However, the reasons for such toxicity have not been elucidated. Here we show that the detrimental effect of excessive Ppz1 expression is due to an increase in its phosphatase activity and not to a plausible down-titration of the PPC decarboxylase components. We have identified several genes encoding ribosomal proteins and ribosome assembly factors as mild high-copy suppressors of the toxic Ppz1 effect. Ppz1 binds to ribosomes engaged in translation and copurifies with diverse ribosomal proteins and translation factors. Ppz1 overexpression results in Gcn2-dependent increased phosphorylation of eIF2α at Ser-51. Consistently, deletion of GCN2 partially suppresses the growth defect of a Ppz1 overexpressing strain. We propose that the deleterious effects of Ppz1 overexpression are in part due to alteration in normal protein synthesis.


Assuntos
Fosfoproteínas Fosfatases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/metabolismo , Saccharomycetales/enzimologia , Saccharomycetales/metabolismo , Carboxiliases , Galactoquinase/metabolismo , Regulação Fúngica da Expressão Gênica , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/toxicidade , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/toxicidade , Saccharomycetales/genética , Transcriptoma
9.
J Dairy Sci ; 103(5): 3912-3923, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32147264

RESUMO

Lipolysis occurs during ripening of dairy products as a result of esterase or lipase activity. Lactic acid bacteria (LAB) are considered to be weakly lipolytic bacteria compared with other species. In cheeses with extended ripening periods, lipolytic LAB may have several advantages. Pediococcus acidilactici is a LAB frequently found in fermented dairy products, but no previous reports exist on their production of esterases or lipases. Our interest in the relationship of LAB and enzymatic characterization is due to the multiple reports of the benefits of LAB in the gut microbiome, particularly at the intestinal membrane. Pediococci have been characterized as probiotic and especially active in membrane interactions. The aim of this project was to purify, characterize, and identify the phosphoesterase produced by P. acidilactici originally isolated from Gouda cheese and determine its phospholipid (PL) hydrolysis profile, with a focus on increased absorption of these compounds in the human gut. Native zymograms were performed to identify a protein with lipolytic activity in the intracellular fraction of P. acidilactici. The enzyme was purified via size-exclusion HPLC, concentrated via ultrafiltration, and identified using sequence analysis in liquid chromatography (LC)-MS/MS. The purified fraction was subjected to biochemical characterization as a function of pH, temperature, ion concentration, hydrolysis of different substrates, and PL. A single protein with a molecular weight of 86 kDa and esterase activity was detected by zymography. Analysis of the LC-MS/MS results identified a putative metallophosphoesterase with a calculated molecular weight of 45.5 kDa, suggesting that this protein is active as a homodimer. The pure protein showed an optimal activity between pH 8.0 to 9.0. The optimal temperature for activity was 37°C, and the enzyme lost 15% of activity after incubation at 90°C for 1 h. This enzyme showed activity on short-chain fatty acids and exhibited high hydrolysis of phosphatidylinositol. It also hydrolyzed phosphatidylserine, phosphatidylcholine, and sphingomyelin. Phosphatidylethanolamine was hydrolyzed but with less efficiency. The characteristics and lipolytic actions exerted by this protein obtained from LAB hold promise for a potential strain of esterase or lipase that may exert human health benefits through increased digestibility and absorption of nutrients found in dairy products.


Assuntos
Queijo/microbiologia , Pediococcus acidilactici/enzimologia , Fosfoproteínas Fosfatases/isolamento & purificação , Animais , Cromatografia Líquida , Humanos , Hidrólise , Lipólise , Peso Molecular , Pediococcus acidilactici/isolamento & purificação , Fosfoproteínas Fosfatases/metabolismo , Espectrometria de Massas em Tandem
10.
Nat Commun ; 11(1): 1176, 2020 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-32132526

RESUMO

Communication by means of diffusible signaling molecules facilitates higher-level organization of cellular populations. Gram-positive bacteria frequently use signaling peptides, which are either detected at the cell surface or 'probed' by intracellular receptors after being pumped into the cytoplasm. While the former type is used to monitor cell density, the functions of pump-probe networks are less clear. Here we show that pump-probe networks can, in principle, perform different tasks and mediate quorum-sensing, chronometric and ratiometric control. We characterize the properties of the prototypical PhrA-RapA system in Bacillus subtilis using FRET. We find that changes in extracellular PhrA concentrations are tracked rather poorly; instead, cells accumulate and strongly amplify the signal in a dose-dependent manner. This suggests that the PhrA-RapA system, and others like it, have evolved to sense changes in the composition of heterogeneous populations and infer the fraction of signal-producing cells in a mixed population to coordinate cellular behaviors.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Bacillus subtilis/fisiologia , Proteínas de Bactérias/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Percepção de Quorum , Transferência Ressonante de Energia de Fluorescência
11.
Artigo em Inglês | MEDLINE | ID: mdl-32005401

RESUMO

As a group of plant-specific proteins, OVATE family protein (OFP) members have been shown to function as transcriptional repressors and involve in plant growth regulation in Arabidopsis and rice. It has also been shown that OFPs can interact with TONNEAU1 Recruiting Motif (TRM) proteins to regulate tomato fruit shape. In this study, we show that mutant plants with knock-down expression of OFP1, OFP2, OFP3, and OFP5 exhibit longer hypocotyls and cotyledons due to enhanced cell elongation. Overexpression of OFPs disturb the arrangement of cortical microtubule arrays in pavement cells and promote abnormal pavement cell expansion perpendicular to the direction of petiole growth, resulting in the kidney-shaped cotyledons in transgenic plants. OFP2 and OFP5 interact directly with the microtubule regulating protein TONNEAU2 (TON2), and genetic analysis suggests TON2 is required for the function of OFPs. We also show that altering the expression of OFPs affects light and BR regulated microtubule reorientation. BR treatment reduce the protein accumulation of OFP2, suggesting OFP2 mediates BR regulated microtubule reorientation. Taken together, our study provides evidences showing that OFP family proteins negatively regulate cell expansion by modulating microtubule reorganization, which requires the function of TON2.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/fisiologia , Crescimento Celular , Microtúbulos/fisiologia , Fosfoproteínas Fosfatases/genética , Proteínas Repressoras/genética , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Proteínas Repressoras/metabolismo
12.
Mol Carcinog ; 59(5): 467-477, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32077156

RESUMO

Protein phosphatase 4 regulatory subunit 1 (PP4R1) has been shown to play a role in the regulation of centrosome maturation, apoptosis, DNA repair, and tumor necrosis factor signaling. However, the function of PP4R1 in non-small-cell lung cancer remains unclear. In this study, we identify PP4R1 as an oncogene through Oncomine database mining and immunohistochemical staining, and we showed that PP4R1 is upregulated in lung cancer tissues as compared with that in normal lung tissues and correlated with a poor prognosis in lung cancer patients. Furthermore, in vitro study by wound-healing and Transwell assay showed that PP4R1 could promote migration and invasion of lung cancer cells. Mechanistic investigations revealed that PP4R1 could cooperate with high mobility group AT-hook 2 and thereby promotes epithelial-mesenchymal transition via MAPK/extracellular receptor kinase activation. Taken together, our study provides a rich resource for understanding PP4R1 in lung cancer and indicates that PP4R1 may serve as a potential biomarker in lung cancer therapies.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/secundário , Movimento Celular , Transição Epitelial-Mesenquimal , Proteína HMGA2/metabolismo , Neoplasias Pulmonares/patologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Apoptose , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Proliferação de Células , Feminino , Seguimentos , Regulação Neoplásica da Expressão Gênica , Proteína HMGA2/genética , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Masculino , Pessoa de Meia-Idade , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/genética , Invasividade Neoplásica , Fosfoproteínas Fosfatases/genética , Prognóstico , Taxa de Sobrevida , Células Tumorais Cultivadas
13.
Dev Cell ; 52(1): 88-103.e18, 2020 01 06.
Artigo em Inglês | MEDLINE | ID: mdl-31910362

RESUMO

After axon outgrowth and synapse formation, the nervous system transitions to a stable architecture. In C. elegans, this transition is marked by the appearance of casein kinase 1δ (CK1δ) in the nucleus. In CK1δ mutants, neurons continue to sprout growth cones into adulthood, leading to a highly ramified nervous system. Nervous system architecture in these mutants is completely restored by suppressor mutations in ten genes involved in transcription termination. CK1δ prevents termination by phosphorylating and inhibiting SSUP-72. SSUP-72 would normally remodel the C-terminal domain of RNA polymerase in anticipation of termination. The antitermination activity of CK1δ establishes the mature state of a neuron by promoting the expression of the long isoform of a single gene, the cytoskeleton protein Ankyrin.


Assuntos
Anquirinas/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Caseína Quinase Idelta/metabolismo , Núcleo Celular/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Transcrição Genética , Animais , Anquirinas/genética , Axônios/fisiologia , Caenorhabditis elegans/genética , Caenorhabditis elegans/crescimento & desenvolvimento , Proteínas de Caenorhabditis elegans/genética , Caseína Quinase Idelta/genética , Núcleo Celular/genética , Fosfoproteínas Fosfatases/genética , Sinapses/fisiologia
14.
FASEB J ; 34(2): 2641-2656, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31909517

RESUMO

PPM1A and PTEN emerged as novel suppressors of chronic kidney disease (CKD). Since loss of PPM1A and PTEN in the tubulointerstitium promotes fibrogenesis, defining molecular events underlying PPM1A/PTEN deregulation is necessary to develop expression rescue as novel therapeutic strategies. Here we identify TGF-ß1 as a principle repressor of PPM1A, as conditional renal tubular-specific induction of TGF-ß1 in mice dramatically downregulates kidney PPM1A expression. TGF-ß1 similarly attenuates PPM1A and PTEN expression in human renal epithelial cells and fibroblasts, via a protein degradation mechanism by promoting their ubiquitination. A proteasome inhibitor MG132 rescues PPM1A and PTEN expression, even in the presence of TGF-ß1, along with decreased fibrogenesis. Restoration of PPM1A or PTEN similarly limits SMAD3 phosphorylation and the activation of TGF-ß1-induced fibrotic genes. Concurrent loss of PPM1A and PTEN levels in aristolochic acid nephropathy further suggests crosstalk between these repressors. PPM1A silencing in renal fibroblasts, moreover, results in PTEN loss, while PTEN stable depletion decreases PPM1A expression with acquisition of a fibroproliferative phenotype in each case. Transient PPM1A expression, conversely, elevates cellular PTEN levels while lentiviral PTEN introduction increases PPM1A expression. PPM1A and PTEN, therefore, co-regulate each other's relative abundance, identifying a previously unknown pathological link between TGF-ß1 repressors, contributing to CKD.


Assuntos
Fibrose/metabolismo , Túbulos Renais/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Proteína Fosfatase 2C/metabolismo , Células Epiteliais/metabolismo , Fibroblastos/metabolismo , Humanos , Rim/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Insuficiência Renal Crônica/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismo
15.
FASEB J ; 34(2): 3179-3196, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31916625

RESUMO

ISOC is a cation current permeating the ISOC channel. In pulmonary endothelial cells, ISOC activation leads to formation of inter-endothelial cell gaps and barrier disruption. The immunophilin FK506-binding protein 51 (FKBP51), in conjunction with the serine/threonine protein phosphatase 5C (PPP5C), inhibits ISOC . Free PPP5C assumes an autoinhibitory state, which has low "basal" catalytic activity. Several S100 protein family members bind PPP5C increasing PPP5C catalytic activity in vitro. One of these family members, S100A6, exhibits a calcium-dependent translocation to the plasma membrane. The goal of this study was to determine whether S100A6 activates PPP5C in pulmonary endothelial cells and contributes to ISOC inhibition by the PPP5C-FKBP51 axis. We observed that S100A6 activates PPP5C to dephosphorylate tau T231. Following ISOC activation, cytosolic S100A6 translocates to the plasma membrane and interacts with the TRPC4 subunit of the ISOC channel. Global calcium entry and ISOC are decreased by S100A6 in a PPP5C-dependent manner and by FKBP51 in a S100A6-dependent manner. Further, calcium entry-induced endothelial barrier disruption is decreased by S100A6 dependent upon PPP5C, and by FKBP51 dependent upon S100A6. Overall, these data reveal that S100A6 plays a key role in the PPP5C-FKBP51 axis to inhibit ISOC and protect the endothelial barrier against calcium entry-induced disruption.


Assuntos
Sinalização do Cálcio , Proteínas de Ciclo Celular/metabolismo , Células Endoteliais/metabolismo , Proteína A6 Ligante de Cálcio S100/metabolismo , Animais , Células Cultivadas , Endotélio Vascular/citologia , Pulmão/irrigação sanguínea , Proteínas Nucleares/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Ligação Proteica , Transporte Proteico , Ratos , Canais de Cátion TRPC/metabolismo , Proteínas de Ligação a Tacrolimo/metabolismo
16.
Elife ; 92020 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-31913126

RESUMO

Alterations involving serine-threonine phosphatase PP2A subunits occur in a range of human cancers, and partial loss of PP2A function contributes to cell transformation. Displacement of regulatory B subunits by the SV40 Small T antigen (ST) or mutation/deletion of PP2A subunits alters the abundance and types of PP2A complexes in cells, leading to transformation. Here, we show that ST not only displaces common PP2A B subunits but also promotes A-C subunit interactions with alternative B subunits (B''', striatins) that are components of the Striatin-interacting phosphatase and kinase (STRIPAK) complex. We found that STRN4, a member of STRIPAK, is associated with ST and is required for ST-PP2A-induced cell transformation. ST recruitment of STRIPAK facilitates PP2A-mediated dephosphorylation of MAP4K4 and induces cell transformation through the activation of the Hippo pathway effector YAP1. These observations identify an unanticipated role of MAP4K4 in transformation and show that the STRIPAK complex regulates PP2A specificity and activity.


Assuntos
Transformação Celular Neoplásica/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas de Ligação a Calmodulina/genética , Proteínas de Ligação a Calmodulina/metabolismo , Proliferação de Células , Feminino , Técnicas de Silenciamento de Genes , Células HEK293 , Xenoenxertos , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Fosfoproteínas Fosfatases/genética , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais , Fatores de Transcrição/metabolismo
17.
Int J Mol Sci ; 21(2)2020 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-31936707

RESUMO

Cells are constantly suffering genotoxic stresses that affect the integrity of our genetic material. Genotoxic insults must be repaired to avoid the loss or inappropriate transmission of the genetic information, a situation that could lead to the appearance of developmental abnormalities and tumorigenesis. To combat this threat, eukaryotic cells have evolved a set of sophisticated molecular mechanisms that are collectively known as the DNA damage response (DDR). This surveillance system controls several aspects of the cellular response, including the detection of lesions, a temporary cell cycle arrest, and the repair of the broken DNA. While the regulation of the DDR by numerous kinases has been well documented over the last decade, the complex roles of protein dephosphorylation have only recently begun to be investigated. Here, we review recent progress in the characterization of DDR-related protein phosphatases during the response to a DNA lesion, focusing mainly on their ability to modulate the DNA damage checkpoint and the repair of the damaged DNA. We also discuss their protein composition and structure, target specificity, and biochemical regulation along the different stages encompassed in the DDR. The compilation of this information will allow us to better comprehend the physiological significance of protein dephosphorylation in the maintenance of genome integrity and cell viability in response to genotoxic stress.


Assuntos
Ciclo Celular/genética , Reparo do DNA/fisiologia , Fosfoproteínas Fosfatases/metabolismo , Animais , Pontos de Checagem do Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Dano ao DNA , Humanos , Proteínas Nucleares , Proteína Fosfatase 1 , Proteína Fosfatase 2 , Proteína Fosfatase 2C , Proteínas Tirosina Fosfatases
18.
FASEB J ; 34(2): 2105-2125, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31908021

RESUMO

How receptor tyrosine kinase (RTK) growth signaling is controlled physiologically is incompletely understood. We have previously provided evidence that the survival and mitotic activities of vascular endothelial cell growth factor receptor-2 (VEGFR2) signaling are dependent on C3a/C5a receptor (C3ar1/C5ar1) and IL-6 receptor (IL-6R)-gp130 joint signaling in a physically interactive platform. Herein, we document that the platelet derived and epidermal growth factor receptors (PDGFR and EGFR) are regulated by the same interconnection and clarify the mechanism underlying the dependence. We show that the joint signaling is required to overcome dominant restraint on RTK function by the combined repression of tonically activated PHLPP, SOCS1/SOCS3, and CK2/Fyn dependent PTEN. Signaling studies showed that augmented PI-3KÉ£ activation is the process that overcomes the multilevel growth restraint. Live-cell flow cytometry and single-particle tracking indicated that blockade of C3ar1/C5ar1 or IL-6R signaling suppresses RTK growth factor binding and RTK complex formation. C3ar1/C5ar1 blockade abrogated growth signaling of four additional RTKs. Active relief of dominant growth repression via joint C3ar1/C5ar1 and IL-6R joint signaling thus enables RTK mitotic/survival signaling.


Assuntos
Células Endoteliais/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Receptor da Anafilatoxina C5a/metabolismo , Receptores de Complemento/metabolismo , Receptores de Interleucina-6/metabolismo , Transdução de Sinais , Proteína 1 Supressora da Sinalização de Citocina/metabolismo , Proteína 3 Supressora da Sinalização de Citocinas/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Linhagem Celular , Células Endoteliais/citologia , Genes Dominantes , Camundongos , Camundongos Knockout , PTEN Fosfo-Hidrolase/genética , Fosfoproteínas Fosfatases/genética , Receptor da Anafilatoxina C5a/genética , Receptores de Complemento/genética , Receptores de Interleucina-6/genética , Proteína 1 Supressora da Sinalização de Citocina/genética , Proteína 3 Supressora da Sinalização de Citocinas/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética
19.
Nat Commun ; 11(1): 138, 2020 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-31919361

RESUMO

In C. elegans, the conserved transcription factor DAF-16/FOXO is a powerful aging regulator, relaying dire conditions into expression of stress resistance and longevity promoting genes. For some of these functions, including low insulin/IGF signaling (IIS), DAF-16 depends on the protein SMK-1/SMEK, but how SMK-1 exerts this role has remained unknown. We show that SMK-1 functions as part of a specific Protein Phosphatase 4 complex (PP4SMK-1). Loss of PP4SMK-1 hinders transcriptional initiation at several DAF-16-activated genes, predominantly by impairing RNA polymerase II recruitment to their promoters. Search for the relevant substrate of PP4SMK-1 by phosphoproteomics identified the conserved transcriptional regulator SPT-5/SUPT5H, whose knockdown phenocopies the loss of PP4SMK-1. Phosphoregulation of SPT-5 is known to control transcriptional events such as elongation and termination. Here we also show that transcription initiating events are influenced by the phosphorylation status of SPT-5, particularly at DAF-16 target genes where transcriptional initiation appears rate limiting, rendering PP4SMK-1 crucial for many of DAF-16's physiological roles.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Fatores de Transcrição Forkhead/genética , Regulação da Expressão Gênica/genética , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Fatores de Elongação da Transcrição/metabolismo , Envelhecimento/genética , Animais , Caenorhabditis elegans/genética , Proteínas Cromossômicas não Histona/genética , Longevidade/genética , Complexos Multiproteicos/metabolismo , Interferência de RNA , RNA Polimerase II/metabolismo , Estresse Fisiológico/genética , Transcrição Genética/genética , Fatores de Elongação da Transcrição/genética
20.
Metabolism ; 103: 154006, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31715176

RESUMO

OBJECTIVE: Glucose and lipid metabolism disorders are a major risk factor for type II diabetes and cardiovascular diseases. Evidence has indicated that the interplay between the liver and adipose tissue is crucial in maintaining energy homeostasis. Recently, the interaction between two distant endocrine organs mainly focuses on the regulation of hormones and receptors. However, as a novel carrier in the inter-tissue communication, exosomes plays a role in liver-fat crosstalk, but its effects on glucose and lipid metabolisms are still unclear. In this study, we sought to investigate the effects of hepatic exosome-derived miR-130a-3p in the regulation of glucose/lipid metabolism in adipose tissues. MEASURE: In vivo, we constructed generalized miR-130a-3p knockout (130KO) and overexpressed (130OE) mice. Wild type (WT), 130KO and 130OE mice (n = 10) were assigned to a randomized controlled trial and were fed diets with either 10% (standard diet, SD) or 60% (high-fat diet, HFD) of total calories from fat (lard). Next, hepatic exosomes were extracted from WT-SD, 130KO-SD and 130OE-SD mice (WT-EXO, KO-EXO, OE-EXO), and 130KO mice were injected with 100 mg hepatic exosomes of different sources via tail-vein (once every 48 h) for 28 days, fed with HFD. In vitro, 3T3-L1 cells were treated with miR-130a-3p mimics, inhibitor and hepatic exosomes. Growth performance and glucose and lipid metabolic profiles were examined. RESULTS: After feeding with HFD, the weights of 130KO mice were markedly higher than WT mice. Over-expression of miR-130a-3p in 130OE mice and intravenous injection of 130OE-EXO in 130KO mice contributed to a positive correlation with the recovery of insulin resistance. In addition, miR-130a-3p mimics and 130OE-EXO treatment of 3T3-L1 cells exhibited decreasing generations of lipid droplets and increasing glucose uptake. Conversely, inhibition of miR-130a-3p in vitro and in vivo resulted in opposite phenotype changes. Furthermore, PHLPP2 was identified as a direct target of miR-130a-3p, and the hepatic exosome-derived miR-130a-3p could improve glucose intolerance via suppressing PHLPP2 to activate AKT-AS160-GLUT4 signaling pathway in adipocytes. CONCLUSIONS: We demonstrated that hepatic exosome-derived miR-130a regulated energy metabolism in adipose tissues, and elucidated a new molecular mechanism that hepatic exosome-derived miR-130a-3p is a crucial participant in organismic energy homeostasis through mediating crosstalk between the liver and adipose tissues.


Assuntos
Adipócitos/metabolismo , Exossomos/metabolismo , Intolerância à Glucose/genética , Fígado/metabolismo , MicroRNAs/genética , Fosfoproteínas Fosfatases/genética , Células 3T3-L1 , Adipogenia/genética , Animais , Regulação para Baixo/genética , Metabolismo Energético/genética , Exossomos/genética , Inativação Gênica , Intolerância à Glucose/metabolismo , Metabolismo dos Lipídeos/genética , Masculino , Camundongos , Camundongos Transgênicos , MicroRNAs/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Transdução de Sinais/genética
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