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1.
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
3.
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
4.
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
5.
Nucleic Acids Res ; 48(9): 4811-4826, 2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32282918

RESUMO

The phosphorylation pattern of Pol2 CTD Y1S2P3T4S5P6S7 repeats comprises an informational code coordinating transcription and RNA processing. cis-trans isomerization of CTD prolines expands the scope of the code in ways that are not well understood. Here we address this issue via analysis of fission yeast peptidyl-prolyl isomerase Pin1. A pin1Δ allele that does not affect growth per se is lethal in the absence of cleavage-polyadenylation factor (CPF) subunits Ppn1 and Swd22 and elicits growth defects absent CPF subunits Ctf1 and Dis2 and termination factor Rhn1. Whereas CTD S2A, T4A, and S7A mutants thrive in combination with pin1Δ, a Y1F mutant does not, nor do CTD mutants in which half the Pro3 or Pro6 residues are replaced by alanine. Phosphate-acquisition genes pho1, pho84 and tgp1 are repressed by upstream lncRNAs and are sensitive to changes in lncRNA 3' processing/termination. pin1Δ hyper-represses PHO gene expression and erases the de-repressive effect of CTD-S7A. Transcriptional profiling delineated sets of 56 and 22 protein-coding genes that are down-regulated and up-regulated in pin1Δ cells, respectively, 77% and 100% of which are downregulated/upregulated when the cis-proline-dependent Ssu72 CTD phosphatase is inactivated. Our results implicate Pin1 as a positive effector of 3' processing/termination that acts via Ssu72.


Assuntos
Regulação Fúngica da Expressão Gênica , Peptidilprolil Isomerase de Interação com NIMA/genética , Fosfoproteínas Fosfatases/genética , Processamento de Terminações 3' de RNA , Proteínas de Schizosaccharomyces pombe/genética , Terminação da Transcrição Genética , Fator de Especificidade de Clivagem e Poliadenilação/genética , Deleção de Genes , Peptidilprolil Isomerase de Interação com NIMA/química , Peptidilprolil Isomerase de Interação com NIMA/metabolismo , Proteínas Nucleares/genética , Fosfatos/metabolismo , Fosforilação , Domínios Proteicos/genética , Pirofosfatases/genética , RNA Polimerase II/genética , RNA-Seq , Regulon , Schizosaccharomyces/enzimologia , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/metabolismo , Serina/metabolismo , Treonina/metabolismo
6.
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
7.
PLoS Genet ; 16(3): e1008317, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32187185

RESUMO

RNA Polymerase II (RNAPII) transcription termination is regulated by the phosphorylation status of the C-terminal domain (CTD). The phosphatase Rtr1 has been shown to regulate serine 5 phosphorylation on the CTD; however, its role in the regulation of RNAPII termination has not been explored. As a consequence of RTR1 deletion, interactions within the termination machinery and between the termination machinery and RNAPII were altered as quantified by Disruption-Compensation (DisCo) network analysis. Of note, interactions between RNAPII and the cleavage factor IA (CF1A) subunit Pcf11 were reduced in rtr1Δ, whereas interactions with the CTD and RNA-binding termination factor Nrd1 were increased. Globally, rtr1Δ leads to decreases in numerous noncoding RNAs that are linked to the Nrd1, Nab3 and Sen1 (NNS) -dependent RNAPII termination pathway. Genome-wide analysis of RNAPII and Nrd1 occupancy suggests that loss of RTR1 leads to increased termination at noncoding genes. Additionally, premature RNAPII termination increases globally at protein-coding genes with a decrease in RNAPII occupancy occurring just after the peak of Nrd1 recruitment during early elongation. The effects of rtr1Δ on RNA expression levels were lost following deletion of the exosome subunit Rrp6, which works with the NNS complex to rapidly degrade a number of noncoding RNAs following termination. Overall, these data suggest that Rtr1 restricts the NNS-dependent termination pathway in WT cells to prevent premature termination of mRNAs and ncRNAs. Rtr1 facilitates low-level elongation of noncoding transcripts that impact RNAPII interference thereby shaping the transcriptome.


Assuntos
Códon de Terminação/genética , RNA Polimerase II/genética , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética , Transcrição Genética/genética , DNA Helicases/genética , Regulação Fúngica da Expressão Gênica/genética , Proteínas Nucleares/genética , Fosfoproteínas Fosfatases/genética , Fosforilação/genética , RNA Helicases/genética , RNA Mensageiro/genética , RNA não Traduzido/genética , Saccharomyces cerevisiae/genética
8.
PLoS One ; 15(3): e0229669, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32163433

RESUMO

Exogenously hypercholesterolemic (ExHC) rats develop diet-induced hypercholesterolemia (DIHC) when fed with dietary cholesterol. Previously, we reported that, under the high-sucrose-diet-feeding condition, a loss-of-function mutation in Smek2 results in low activity of fatty acid synthase (FAS) followed by the shortage of hepatic triacylglycerol content in ExHC rats and the onset of DIHC. However, the relationship between the Smek2 mutation and FAS dysfunction is still unclear. Here, we focused on carbohydrate metabolism, which provides substrates for FAS, and analyzed carbohydrate and lipid metabolisms in ExHC rats to clarify how the deficit of Smek2 causes DIHC. Male ExHC and SD rats were fed high-sucrose or high-starch diets containing 1% cholesterol for 2 weeks. Serum cholesterol levels of the ExHC rats were higher, regardless of the dietary carbohydrate. Hepatic triacylglycerol levels were higher in only the SD rats fed the high-sucrose diet. Moreover, the ExHC rats exhibited a diabetes-like status and accumulation of hepatic glycogen and low hepatic mRNA levels of liver-type phosphofructokinase (Pfkl), which encodes a rate-limiting enzyme for glycolysis. These results suggest that the glucose utilization, particularly glycolysis, is impaired in the liver of ExHC rats. To evaluate how the diet with extremely low glucose affect to DIHC, ExHC.BN-Dihc2BN, a congenic strain that does not develop DIHC, and ExHC rats were fed a high-fructose diet containing 1% cholesterol for 2 weeks. The serum cholesterol and hepatic triacylglycerol levels were similar in the strains. Results of water-soluble metabolite analysis with primary hepatocytes, an increase in fructose-6-phosphate and decreases in succinate, malate and aspartate in ExHC rats, support impaired glycolysis in the ExHC rats. Thus, the Smek2 mutation causes abnormal hepatic glucose utilization via downregulation of Pfkl expression. This abnormal glucose metabolism disrupts hepatic fatty acid synthesis and causes DIHC in the ExHC rats.


Assuntos
Glucose/metabolismo , Hipercolesterolemia/etiologia , Hipercolesterolemia/metabolismo , Fígado/metabolismo , Animais , Animais Congênicos , Colesterol na Dieta/administração & dosagem , Colesterol na Dieta/efeitos adversos , Modelos Animais de Doenças , Regulação para Baixo , Ácido Graxo Sintase Tipo I/metabolismo , Glicólise/genética , Hipercolesterolemia/genética , Mutação com Perda de Função , Masculino , Fosfofrutoquinases/genética , Fosfofrutoquinases/metabolismo , Fosfoproteínas Fosfatases/deficiência , Fosfoproteínas Fosfatases/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Endogâmicos BN , Ratos Sprague-Dawley
9.
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
10.
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
11.
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
12.
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
13.
Biochem J ; 477(2): 431-444, 2020 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-31904830

RESUMO

Protein Ser/Thr phosphatase-6 (PP6) regulates pathways for activation of NF-kB, YAP1 and Aurora A kinase (AURKA). PP6 is a heterotrimer comprised of a catalytic subunit, one of three different SAPS subunits and one of three different ankyrin-repeat ANKRD subunits. Here, we show FLAG-PP6C expressed in cells preferentially binds endogenous SAPS3, and the complex is active with the chemical substrate DiFMUP. SAPS3 has multiple acidic sequence motifs recognized by protein kinase CK2 (CK2) and SAPS3 is phosphorylated by purified CK2, without affecting its associated PP6 phosphatase activity. However, HA3-SAPS3-PP6 phosphatase activity using pT288 AURKA as substrate is significantly increased by phosphorylation with CK2. The substitution of Ala in nine putative phosphorylation sites in SAPS3 was required to prevent CK2 activation of the phosphatase. Different CK2 chemical inhibitors equally increased phosphorylation of endogenous AURKA in living cells, consistent with reduction in PP6 activity. CRISPR/Cas9 deletion or siRNA knockdown of SAPS3 resulted in highly activated endogenous AURKA, and a high proportion of cells with abnormal nuclei. Activation of PP6 by CK2 can form a feedback loop with bistable changes in substrates.


Assuntos
Aurora Quinase A/genética , Caseína Quinase II/química , Fosfoproteínas Fosfatases/genética , Alanina/genética , Substituição de Aminoácidos/genética , Aurora Quinase A/química , Sistemas CRISPR-Cas/genética , Caseína Quinase II/genética , Domínio Catalítico/genética , Inibidores Enzimáticos/farmacologia , Células HeLa , Humanos , Fosfoproteínas Fosfatases/antagonistas & inibidores , Fosfoproteínas Fosfatases/química , Fosforilação/genética , Ligação Proteica/efeitos dos fármacos , RNA Interferente Pequeno/genética , Especificidade por Substrato/efeitos dos fármacos
14.
Int J Mol Sci ; 21(2)2020 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-31936296

RESUMO

Eukaryotic cells make the decision to proliferate, to differentiate or to cease dividing during G1, before passage through the restriction point or Start. Keeping cyclin-dependent kinase (CDK) activity low during this period restricts commitment to a new cell cycle and is essential to provide the adequate timeframe for the sensing of environmental signals. Here, we review the role of protein phosphatases in the modulation of CDK activity and as the counteracting force for CDK-dependent substrate phosphorylation, in budding and fission yeast. Moreover, we discuss recent findings that place protein phosphatases in the interface between nutritional signalling pathways and the cell cycle machinery.


Assuntos
Quinases Ciclina-Dependentes/genética , Fase G1/genética , Fosfoproteínas Fosfatases/genética , Proliferação de Células/genética , Fosforilação , Saccharomyces cerevisiae/genética , Transdução de Sinais/genética
15.
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
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.
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
18.
Plant Mol Biol ; 102(1-2): 199-212, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31813113

RESUMO

KEY MESSAGE: The kinase-associated protein phosphatase, KAPP, is negatively involved in abscisic acid (ABA) signaling. KAPP interacts physically with SnRK2.2, SnRK2.3 and SnRK2.6, and functionally acts upstream of SnRK2.2 and SnRK2.3. The kinase-associated protein phosphatase (KAPP) has been reported to be involved in the regulation of many developmental and signaling events, but it remains unknown whether KAPP is involved in ABA signaling. Here, we report that KAPP is negatively involved in ABA-mediated seed germination and early seedling growth in Arabidopsis thaliana. The two loss-of-function mutants of KAPP, kapp-1 and kapp-2, exhibit increased ABA sensitivity in ABA-induced seed germination inhibition and post-germination growth arrest. The three closely-related protein kinase, (SNF1)-related protein kinase SnRK2.2, SnRK2.3 and SnRK2.6, which play critical roles in ABA signaling, interact and co-localize with KAPP. Genetic evidence showed that the ABA-hypersensitive phenotypes caused by KAPP mutation were suppressed by the double mutation of SnRK2.2 and SnRK2.3, indicating that KAPP functions upstream of SnRK2.2 and SnRK2.3 in ABA signaling. RNA-sequencing analysis revealed that KAPP mutation affects expression of multiple ABA-responsive genes. These results demonstrated that KAPP is negatively involved in plant response to ABA, which help to understand the complicated ABA signaling mechanism.


Assuntos
Ácido Abscísico/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Germinação , Mutação , Fenótipo , Fosfoproteínas Fosfatases/genética , Folhas de Planta/metabolismo , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/genética , Plântula/crescimento & desenvolvimento , Análise de Sequência , Análise de Sequência de RNA , Transdução de Sinais , Estresse Fisiológico , Tabaco/genética
19.
J Appl Microbiol ; 128(2): 606-617, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31606917

RESUMO

AIMS: The object of this study was to identify potential health concerns of the Aeromons spp. isolated from marketed Pacific abalone (Haliotis discus hannai) with respect to their virulence and antimicrobial resistance patterns. METHODS AND RESULTS: We identified 29 strains of aeromonads consisting of five species; Aeromonas hydrophila (n = 9), Aeromonas enteropelogenes (n = 14), Aeromonas veronii (n = 3), Aeromonas salmonicida (n = 2) and Aeromonas sobria (n = 1), by employing series of biochemical tests and gene sequencing. In the phenotypic virulence assays, all isolates showed gelatinase and caseinase activities, while lipase formation (69%), phospholipase production (90%), DNase formation (82%), slime production (49%) and haemolysis activity (α = 18% and ß = 82%) were also detected among isolates. Prevalence of virulence genes; aerA (100%), fla (66%), ahyB (73%), act (52%), alt (42%), ast (35%), ser (52%), gcat (69%), ascV (43%), hlyA (83%), lip (52%) and exu (59%) were detected by PCR assays. In disc diffusion test, 100% resistance was detected against ampicillin while cephalothin, rifampicin, oxytetracycline, colistine sulphate, nalidixic acid and piperaciliin were resisted by 86, 73, 42, 35, 28, 20 and 20% of the isolates respectively. Thirteen (45%) of the isolates showed multiple antimicrobial resistance (MAR) indices ≥ 0·2. CONCLUSIONS: Our findings suggest that the potential health risk posed by the abalone-borne Aeromonas spp. should not be underestimated. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first time to evaluate possible public health risks upon consumption of abalone harbored Aeromonas spp. and also to isolate potential pathogenic and multidrug-resistant Aeromonas spp. from Pacific abalone in Korea.


Assuntos
Aeromonas/efeitos dos fármacos , Aeromonas/isolamento & purificação , Farmacorresistência Bacteriana , Gastrópodes/microbiologia , Alimentos Marinhos/microbiologia , Aeromonas/classificação , Aeromonas/genética , Animais , Antibacterianos/farmacologia , Contaminação de Alimentos/análise , Ácido Nalidíxico/farmacologia , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Reação em Cadeia da Polimerase , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , República da Coreia
20.
Proc Natl Acad Sci U S A ; 117(1): 328-336, 2020 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-31843888

RESUMO

The fundamental importance of the 26S proteasome in health and disease suggests that its function must be finely controlled, and yet our knowledge about proteasome regulation remains limited. Posttranslational modifications, especially phosphorylation, of proteasome subunits have been shown to impact proteasome function through different mechanisms, although the vast majority of proteasome phosphorylation events have not been studied. Here, we have characterized 1 of the most frequently detected proteasome phosphosites, namely Ser361 of Rpn1, a base subunit of the 19S regulatory particle. Using a variety of approaches including CRISPR/Cas9-mediated gene editing and quantitative mass spectrometry, we found that loss of Rpn1-S361 phosphorylation reduces proteasome activity, impairs cell proliferation, and causes oxidative stress as well as mitochondrial dysfunction. A screen of the human kinome identified several kinases including PIM1/2/3 that catalyze S361 phosphorylation, while its level is reversibly controlled by the proteasome-resident phosphatase, UBLCP1. Mechanistically, Rpn1-S361 phosphorylation is required for proper assembly of the 26S proteasome, and we have utilized a genetic code expansion system to directly demonstrate that S361-phosphorylated Rpn1 more readily forms a precursor complex with Rpt2, 1 of the first steps of 19S base assembly. These findings have revealed a prevalent and biologically important mechanism governing proteasome formation and function.


Assuntos
Proteínas de Membrana/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Subunidades Proteicas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Sistemas CRISPR-Cas/genética , Linhagem Celular , Ensaios Enzimáticos , Técnicas de Introdução de Genes , Humanos , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Mitocôndrias/metabolismo , Proteínas Nucleares/genética , Estresse Oxidativo , Fosfoproteínas Fosfatases/genética , Fosforilação/fisiologia , Complexo de Endopeptidases do Proteassoma/genética , Subunidades Proteicas/genética , Proteínas Serina-Treonina Quinases/genética , RNA Interferente Pequeno/metabolismo , Serina/metabolismo , Transativadores/genética , Transativadores/metabolismo
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