RESUMEN
Influenza A viruses (IAVs) are respiratory pathogens that are able to hijack multiple cellular mechanisms to drive their replication. Consequently, several viral and cellular proteins undergo posttranslational modifications such as dynamic phosphorylation/dephosphorylation. In eukaryotic cells, dephosphorylation is mainly catalyzed by protein phosphatase 2A (PP2A). While the function of kinases in IAV infection is quite well studied, only little is known about the role of PP2A in IAV replication. Here, we show, by using knockdown and inhibition approaches of the catalytic subunit PP2Ac, that this phosphatase is important for efficient replication of several IAV subtypes. This could neither be attributed to alterations in the antiviral immune response nor to changes in transcription or translation of viral genes. Interestingly, decreased PP2Ac levels resulted in a significantly reduced cell viability after IAV infection. Comprehensive kinase activity profiling identified an enrichment of process networks related to apoptosis and indicated a synergistic action of hyper-activated PI3K/Akt, MAPK/JAK-STAT and NF-kB signaling pathways, collectively resulting in increased cell death. Taken together, while IAV seems to effectively tap leftover PP2A activity to ensure efficient viral replication, reduced PP2Ac levels fail to orchestrate cell survival mechanisms to protect infected cells from early cell death.
Asunto(s)
Apoptosis , Supervivencia Celular , Virus de la Influenza A/fisiología , Gripe Humana/inmunología , Gripe Humana/virología , Infecciones por Orthomyxoviridae/virología , Proteína Fosfatasa 2/fisiología , Células A549 , Animales , Línea Celular , Perros , Técnicas de Silenciamiento del Gen , Interacciones Microbiota-Huesped , Humanos , Células de Riñón Canino Madin Darby , Fosforilación , Transducción de Señal , Replicación ViralRESUMEN
Chromosome instability (CIN) is a major hallmark of cancer cells and believed to drive tumor progression. Several cellular defects including weak centromeric cohesion are proposed to promote CIN, but the molecular mechanisms underlying these defects are poorly understood. In a screening for SET protein levels in various cancer cell lines, we found that most of the cancer cells exhibit higher SET protein levels than nontransformed cells, including RPE-1. Cancer cells with elevated SET often show weak centromeric cohesion, revealed by MG132-induced cohesion fatigue. Partial SET knockdown largely strengthens centromeric cohesion in cancer cells without increasing overall phosphatase 2A (PP2A) activity. Pharmacologically increased PP2A activity in these cancer cells barely ameliorates centromeric cohesion. These results suggest that compromised PP2A activity, a common phenomenon in cancer cells, may not be responsible for weak centromeric cohesion. Furthermore, centromeric cohesion in cancer cells can be strengthened by ectopic Sgo1 overexpression and weakened by SET WT, not by Sgo1-binding-deficient mutants. Altogether, these findings demonstrate that SET overexpression contributes to impaired centromeric cohesion in cancer cells and illustrate misregulated SET-Sgo1 pathway as an underlying mechanism.
Asunto(s)
Inestabilidad Cromosómica/fisiología , Segregación Cromosómica/genética , Proteínas de Unión al ADN/metabolismo , Chaperonas de Histonas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Línea Celular Tumoral , Centrómero/fisiología , Proteínas Cromosómicas no Histona/metabolismo , Segregación Cromosómica/fisiología , Proteínas de Unión al ADN/fisiología , Chaperonas de Histonas/fisiología , Humanos , Mitosis , Proteínas Nucleares/metabolismo , Proteína Fosfatasa 2/metabolismo , Proteína Fosfatasa 2/fisiología , Proteínas Serina-Treonina Quinasas/metabolismoRESUMEN
The serine-threonine protein phosphatase 2A (PP2A) is a heterotrimeric enzyme complex that regulates many fundamental cellular processes. PP2A is involved in tumorigenesis because mutations in the scaffold subunit, PPP2R1B, were found in several types of cancers. However, the biological function of PPP2R1B remains largely unknown. We report here that homozygous deletion of Ppp2r1b in Mus musculus impairs meiotic recombination and causes meiotic arrest in spermatocytes. Consistently, male mice lacking Ppp2r1b are characterized with infertility. Furthermore, heterozygous missense mutations in the Homo sapiens PPP2R1B gene, which encodes PPP2R1B, are identified in azoospermia patients with meiotic arrest. We found that PPP2R1B mutants are susceptible to degradation by an E3 ligase CRL4ADCAF6 , and resistant to de-polyubiquitylation by ubiquitin-specific protease 5 (USP5). In addition, heterozygous mutations in PPP2R1B reduce stability of the wild-type PPP2R1B. Our results demonstrate an essential role of PPP2R1B in spermatogenesis and identify upstream regulators of PPP2R1B.
Asunto(s)
Infertilidad Masculina/patología , Mutación , Proteína Fosfatasa 2/metabolismo , Proteína Fosfatasa 2/fisiología , Espermatogénesis , Testículo/patología , Ubiquitinación , Animales , Familia , Femenino , Homocigoto , Humanos , Infertilidad Masculina/etiología , Infertilidad Masculina/metabolismo , Masculino , Meiosis , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína Fosfatasa 2/genética , Testículo/metabolismoRESUMEN
The protein phosphatase PP2A is essential for the control of integrated eukaryotic cell functioning. Several cellular and developmental events, e.g., plant growth regulator (PGR) mediated signaling pathways are regulated by reversible phosphorylation of vesicle traffic proteins. Reviewing present knowledge on the relevant role of PP2A is timely. We discuss three aspects: (1) PP2A regulates microtubule-mediated vesicle delivery during cell plate assembly. PP2A dephosphorylates members of the microtubule associated protein family MAP65, promoting their binding to microtubules. Regulation of phosphatase activity leads to changes in microtubule organization, which affects vesicle traffic towards cell plate and vesicle fusion to build the new cell wall between dividing cells. (2) PP2A-mediated inhibition of target of rapamycin complex (TORC) dependent signaling pathways contributes to autophagy and this has possible connections to the brassinosteroid signaling pathway. (3) Transcytosis of vesicles transporting PIN auxin efflux carriers. PP2A regulates vesicle localization and recycling of PINs related to GNOM (a GTP-GDP exchange factor) mediated pathways. The proper intracellular traffic of PINs is essential for auxin distribution in the plant body, thus in whole plant development. Overall, PP2A has essential roles in membrane interactions of plant cell and it is crucial for plant development and stress responses.
Asunto(s)
Vesículas Citoplasmáticas/metabolismo , Desarrollo de la Planta/fisiología , Proteína Fosfatasa 2/fisiología , Transporte Biológico/genética , Fosforilación/genética , Células Vegetales/metabolismo , Desarrollo de la Planta/genética , Proteína Fosfatasa 2/genética , Transducción de Señal/fisiologíaRESUMEN
Loss of hepatic fructose-1, 6-bisphosphate aldolase B (Aldob) leads to a paradoxical up-regulation of glucose metabolism to favor hepatocellular carcinogenesis (HCC), but the upstream signaling events remain poorly defined. Akt is highly activated in HCC, and targeting Akt is being explored as a potential therapy for HCC. Herein, we demonstrate that Aldob suppresses Akt activity and tumor growth through a protein complex containing Aldob, Akt, and protein phosphatase 2A (PP2A), leading to inhibition of cell viability, cell cycle progression, glucose uptake, and metabolism. Interestingly, Aldob directly interacts with phosphorylated Akt (p-Akt) and promotes the recruitment of PP2A to dephosphorylate p-Akt, and this scaffolding effect of Aldob is independent of its enzymatic activity. Loss of Aldob or disruption of Aldob/Akt interaction in Aldob R304A mutant restores Akt activity and tumor-promoting effects. Consistently, Aldob and p-Akt expression are inversely correlated in human HCC tissues, and Aldob down-regulation coupled with p-Akt up-regulation predicts a poor prognosis for HCC. We have further discovered that Akt inhibition or a specific small-molecule activator of PP2A (SMAP) efficiently attenuates HCC tumorigenesis in xenograft mouse models. Our work reveals a novel nonenzymatic role of Aldob in negative regulation of Akt activation, suggesting that directly inhibiting Akt activity or through reactivating PP2A may be a potential therapeutic approach for HCC treatment.
Asunto(s)
Carcinoma Hepatocelular/metabolismo , Fructosa-Bifosfato Aldolasa/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Animales , Apoptosis/efectos de los fármacos , Carcinoma Hepatocelular/fisiopatología , Línea Celular Tumoral , Supervivencia Celular/genética , China , Fructosa-Bifosfato Aldolasa/biosíntesis , Fructosa-Bifosfato Aldolasa/genética , Glucosa/metabolismo , Humanos , Neoplasias Hepáticas/metabolismo , Masculino , Ratones , Ratones Desnudos , Fosforilación , Proteína Fosfatasa 2/metabolismo , Proteína Fosfatasa 2/fisiología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Protein Phosphatase 2A (PP2A) is a heterotrimer composed of scaffolding (A), catalytic (C), and regulatory (B) subunits. PP2A complexes with B56 subunits are targeted by Shugoshin and BUBR1 to protect centromeric cohesion and stabilise kinetochore-microtubule attachments in yeast and mouse meiosis. In Caenorhabditis elegans, the closest BUBR1 orthologue lacks the B56-interaction domain and Shugoshin is not required for meiotic segregation. Therefore, the role of PP2A in C. elegans female meiosis is unknown. We report that PP2A is essential for meiotic spindle assembly and chromosome dynamics during C. elegans female meiosis. BUB-1 is the main chromosome-targeting factor for B56 subunits during prometaphase I. BUB-1 recruits PP2A:B56 to the chromosomes via a newly identified LxxIxE motif in a phosphorylation-dependent manner, and this recruitment is important for proper chromosome congression. Our results highlight a novel mechanism for B56 recruitment, essential for recruiting a pool of PP2A involved in chromosome congression during meiosis I.
Asunto(s)
Proteínas de Caenorhabditis elegans/fisiología , Caenorhabditis elegans/fisiología , Cromosomas/fisiología , Meiosis/fisiología , Oocitos/fisiología , Proteína Fosfatasa 2/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Animales , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Segregación Cromosómica , Oocitos/metabolismo , Proteína Fosfatasa 2/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismoRESUMEN
Insulin and insulin-like growth factors are longevity determinants that negatively regulate Forkhead box class O (FoxO) transcription factors. In C. elegans mutations that constitutively activate DAF-16, the ortholog of mammalian FoxO3a, extend lifespan by two-fold. While environmental insults induce DAF-16 activity in younger animals, it also becomes activated in an age-dependent manner in the absence of stress, modulating gene expression well into late adulthood. The mechanism by which DAF-16 activity is regulated during aging has not been defined. Since phosphorylation of DAF-16 generally leads to its inhibition, we asked whether phosphatases might be necessary for its increased transcriptional activity in adult C. elegans. We focused on the PP2A/4/6 subfamily of phosphoprotein phosphatases, members of which had been implicated to regulate DAF-16 under low insulin signaling conditions but had not been investigated during aging in wildtype animals. Using reverse genetics, we functionally characterized all C. elegans orthologs of human catalytic, regulatory, and scaffolding subunits of PP2A/4/6 holoenzymes in postreproductive adults. We found that PP2A complex constituents PAA-1 and PPTR-1 regulate DAF-16 transcriptional activity during aging and that they cooperate with the catalytic subunit LET-92 to protect adult animals from ultraviolet radiation. PP4 complex members PPH-4.1/4.2, and SMK-1 also appear to regulate DAF-16 in an age-dependent manner, and together with PPFR-2 they contribute to innate immunity. Interestingly, SUR-6 but no other subunit of the PP2A complex was necessary for the survival of pathogen-infected animals. Finally, we found that PP6 complex constituents PPH-6 and SAPS-1 contribute to host defense during aging, apparently without affecting DAF-16 transcriptional activity. Our studies indicate that a set of PP2A/4/6 complexes protect adult C. elegans from environmental stress, thus preserving healthspan. Therefore, along with their functions in cell division and development, the PP2A/4/6 phosphatases also appear to play critical roles later in life.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Factores de Transcripción Forkhead/metabolismo , Proteína Fosfatasa 2/metabolismo , Estrés Fisiológico/fisiología , Envejecimiento/metabolismo , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/fisiología , Factores de Transcripción Forkhead/fisiología , Longevidad/genética , Fosfoproteínas Fosfatasas/metabolismo , Fosforilación , Proteína Fosfatasa 2/fisiología , Transducción de SeñalRESUMEN
BACKGROUND: Hepatocellular carcinoma (HCC) is the major type of primary liver cancer. Mice lacking the tumor-suppressive protein phosphatase 2A subunit B56δ (Ppp2r5d) spontaneously develop HCC, correlating with increased c-MYC oncogenicity. MATERIALS AND METHODS: We used two-dimensional difference gel electrophoresis-coupled matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to identify differential proteomes of livers from wild-type, non-cancerous and HCC-affected B56δ knockout mice. RESULTS: A total of 23 proteins were differentially expressed/regulated in liver between wild-type and non-cancerous knockout mice, and 119 between non-cancerous and HCC knockout mice ('cancer proteins'). Overlap with our reported differential transcriptome data was poor. Overall, 56% of cancer proteins were reported before in HCC proteomics studies; 44% were novel. Gene Ontology analysis revealed cancer proteins mainly associated with liver metabolism (18%) and mitochondria (15%). Ingenuity Pathway Analysis identified 'cancer' and 'gastrointestinal disease' as top hits. CONCLUSION: We identified several proteins for further exploration as novel potential HCC biomarkers, and independently underscored the relevance of Ppp2r5d knockout mice as a valuable hepatocarcinogenesis model.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Proteína Fosfatasa 2/fisiología , Proteoma/análisis , Proteoma/metabolismo , Animales , Apoptosis , Carcinoma Hepatocelular/patología , Proliferación Celular , Neoplasias Hepáticas/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Células Tumorales CultivadasRESUMEN
Purpose: Intrinsically photosensitive retinal ganglion cells (ipRGCs) that express the visual pigment melanopsin regulate non-image-forming visual tasks, such as circadian photoentrainment and pupil constriction, as well as contrast detection for image formation. Sustained ipRGC function throughout the day is, therefore, of great importance. Melanopsin is a bistable rhabdomeric-type (R-type) visual pigment, which is thought to use light to regenerate its chromophore from all-trans-retinal back to 11-cis-retinal and does not depend on constant chromophore supply to the extent required by visual pigment in rod and cone photoreceptors. Like the majority of photopigments and G-protein-coupled receptors (GPCRs), melanopsin deactivation requires C-terminal phosphorylation and subsequent ß-arrestin binding. We hypothesize that melanopsin utilizes canonical GPCR resensitization mechanisms, including dephosphorylation and endocytosis, during the light, and together, they provide a mechanism for prolonged light responses. Methods: Here, we examined expression of protein phosphatases from a variety of subfamilies by RT-PCR and immunohistochemical analyses of the mouse retina. The expression of protein phosphatase 2A (PP2A) in ipRGCs was assessed. We also examine the role of phosphatase and endocytic activity in sustaining melanopsin signaling using transiently-transfected HEK293 cells. Results: Our analyses suggest that melanopsin-mediated light responses can be rapidly and extensively enhanced by PP2A activity. Light-activated melanopsin undergoes endocytosis in a clathrin-dependent manner. This endocytic activity enhances light responses upon repeated stimulation, implicating a role for endocytic activity in resensitization. Conclusions: Thus, we propose that melanopsin phototransduction is maintained by utilizing canonical GPCR resensitization mechanisms rather than reliance on chromophore replenishment from supporting cells.
Asunto(s)
Clatrina/fisiología , Endocitosis/fisiología , Regulación de la Expresión Génica/fisiología , Proteína Fosfatasa 2/fisiología , Células Ganglionares de la Retina/metabolismo , Opsinas de Bastones/metabolismo , Animales , Señalización del Calcio/fisiología , Células HEK293 , Humanos , Ratones , Ratones Endogámicos C57BL , Microscopía Confocal , Estimulación Luminosa , Plásmidos , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Células Ganglionares de la Retina/efectos de la radiación , Transfección , Visión Ocular/fisiologíaRESUMEN
Protein phosphatase 2A is a ubiquitously expressed serine/threonine phosphatase that comprises a scaffold, a catalytic, and multiple regulatory subunits and has been shown to be important in the expression of autoimmunity. We considered that a distinct subunit may account for the decreased production of IL-2 in people and mice with systemic autoimmunity. We show that the regulatory subunit PPP2R2D is increased in T cells from people with systemic lupus erythematosus and regulates IL-2 production. Mice lacking PPP2R2D only in T cells produce more IL-2 because the IL-2 gene and genes coding for IL-2-enhancing transcription factors remain open, while the levels of the enhancer phosphorylated CREB are high. Mice with T cell-specific PPP2R2D deficiency display less systemic autoimmunity when exposed to a TLR7 stimulator. While genes related to Treg function do not change in the absence of PPP2R2D, Tregs exhibit high suppressive function in vitro and in vivo. Because the ubiquitous expression of protein phosphatase 2A cannot permit systemic therapeutic manipulation, the identification of regulatory subunits able to control specific T cell functions opens the way for the development of novel, function-specific drugs.
Asunto(s)
Autoinmunidad , Interleucina-2/metabolismo , Lupus Eritematoso Sistémico/patología , Proteína Fosfatasa 2/metabolismo , Proteína Fosfatasa 2/fisiología , Linfocitos T Reguladores/inmunología , Adulto , Animales , Estudios de Casos y Controles , Femenino , Humanos , Lupus Eritematoso Sistémico/genética , Lupus Eritematoso Sistémico/inmunología , Lupus Eritematoso Sistémico/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Fosforilación , Proteína Fosfatasa 2/genética , Adulto JovenRESUMEN
Protein phosphatase 2A (PP2A) enzymes can suppress tumors, but they are often inactivated in human cancers overexpressing inhibitory proteins. Here, we identify a class of small-molecule iHAPs (improved heterocyclic activators of PP2A) that kill leukemia cells by allosterically assembling a specific heterotrimeric PP2A holoenzyme consisting of PPP2R1A (scaffold), PPP2R5E (B56ε, regulatory), and PPP2CA (catalytic) subunits. One compound, iHAP1, activates this complex but does not inhibit dopamine receptor D2, a mediator of neurologic toxicity induced by perphenazine and related neuroleptics. The PP2A complex activated by iHAP1 dephosphorylates the MYBL2 transcription factor on Ser241, causing irreversible arrest of leukemia and other cancer cells in prometaphase. In contrast, SMAPs, a separate class of compounds, activate PP2A holoenzymes containing a different regulatory subunit, do not dephosphorylate MYBL2, and arrest tumor cells in G1 phase. Our findings demonstrate that small molecules can serve as allosteric switches to activate distinct PP2A complexes with unique substrates.
Asunto(s)
Proteína Fosfatasa 2/metabolismo , Apoptosis , Proteínas de Ciclo Celular/efectos de los fármacos , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Activadores de Enzimas/metabolismo , Fase G1 , Humanos , Complejos Multiproteicos/metabolismo , Complejos Multiproteicos/fisiología , Fenotiazinas/farmacología , Fosforilación , Proteína Fosfatasa 2/fisiología , Subunidades de Proteína/metabolismo , Transactivadores/efectos de los fármacos , Transactivadores/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Cadmium (Cd) is phytotoxic and detoxified primarily via phytochelatin (PC) complexation in Arabidopsis. Here, we explore Cd toxicity responses and defence mechanisms beyond the PC pathway using forward genetics approach. We isolated an Arabidopsis thaliana Cd-hypersensitive mutant, Cd-induced short root 1 (cdsr1) in the PC synthase mutant (cad1-3) background. Using genomic resequencing and complementation, we identified PP2A-4C as the causal gene for the mutant phenotype, which encodes a catalytic subunit of protein phosphatase 2A (PP2A). Root and shoot growth of cdsr1 cad1-3 and cdsr1 were more sensitive to Cd than their respective wild-type cad1-3 and Col-0. A mutant of the PP2A scaffolding subunit 1A was also more sensitive to Cd. PP2A-4C was localized in the cytoplasm and nucleus and PP2A-4C expression was downregulated by Cd in cad1-3. PP2A enzyme activity was decreased in cdsr1 and cdsr1 cad1-3 under Cd stress. The expression of 1-aminocyclopropane-1-carboxylic acid synthase genes ACS2 and ACS6 was upregulated by Cd more in cad1-3 and cdsr1 cad1-3 than in Col-0 and the double mutant had a higher ACS activity. cdsr1 cad1-3 and cdsr1 overproduced ethylene under Cd stress. The results suggest that PP2A containing 1A and 4C subunits alleviates Cd-induced growth inhibition by modulating ethylene production.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Cadmio/toxicidad , Etilenos/biosíntesis , Proteína Fosfatasa 2/metabolismo , Arabidopsis/efectos de los fármacos , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/fisiología , Clonación Molecular , Regulación de la Expresión Génica de las Plantas , Genes de Plantas/genética , Raíces de Plantas/metabolismo , Brotes de la Planta/metabolismo , Proteína Fosfatasa 2/genética , Proteína Fosfatasa 2/fisiología , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Mutations in LRRK2 are currently recognized as the most common monogenetic cause of Parkinsonism. The elevation of kinase activity of LRRK2 that frequently accompanies its mutations is widely thought to contribute to its toxicity. Accordingly, many groups have developed LRRK2-specific kinase inhibitors as a potential therapeutic strategy. Given that protein phosphorylation is a reversible event, we sought to elucidate the phosphatase(s) that can reverse LRRK2-mediated phosphorylation, with the view that targeting this phosphatase(s) may similarly be beneficial. Using an unbiased RNAi phosphatase screen conducted in a Drosophila LRRK2 model, we identified PP2A as a genetic modulator of LRRK2-induced neurotoxicity. Further, we also identified ribosomal S6 kinase (S6K), a target of PP2A, as a novel regulator of LRRK2 function. Finally, we showed that modulation of PP2A or S6K activities ameliorates LRRK2-associated disease phenotype in Drosophila.
Asunto(s)
Proteínas de Drosophila/genética , Proteínas de Drosophila/fisiología , Drosophila melanogaster/enzimología , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/genética , Proteína Fosfatasa 2/fisiología , Proteínas Quinasas S6 Ribosómicas/fisiología , Animales , Animales Modificados Genéticamente , Línea Celular , Ceramidas/farmacología , Modelos Animales de Enfermedad , Proteínas de Drosophila/antagonistas & inhibidores , Proteínas de Drosophila/metabolismo , Evaluación Preclínica de Medicamentos , Activación Enzimática/efectos de los fármacos , Clorhidrato de Fingolimod/farmacología , Mutación con Ganancia de Función , Técnicas de Silenciamiento del Gen , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Actividad Motora/efectos de los fármacos , Mutación Missense , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Fosfoproteínas Fosfatasas/genética , Fosfoproteínas Fosfatasas/fisiología , Fosforilación/efectos de los fármacos , Proteína Fosfatasa 2/antagonistas & inhibidores , Proteína Fosfatasa 2/genética , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Interferencia de ARN , ARN Interferente Pequeño/genética , Proteínas Recombinantes/metabolismo , Proteínas Quinasas S6 Ribosómicas/antagonistas & inhibidores , Proteínas Quinasas S6 Ribosómicas/genética , Proteínas Quinasas S6 Ribosómicas 90-kDa/antagonistas & inhibidores , Proteínas Quinasas S6 Ribosómicas 90-kDa/metabolismoRESUMEN
Across most sexually reproducing animals, centrosomes are provided to the oocyte through fertilization and must be positioned properly to establish the zygotic mitotic spindle. How centrosomes are positioned in space and time through the concerted action of key mitotic entry biochemical regulators, including protein phosphatase 2A (PP2A-B55/SUR-6), biophysical regulators, including dynein, and the nuclear lamina is unclear. Here, we uncover a role for PP2A-B55/SUR-6 in regulating centrosome separation. Mechanistically, PP2A-B55/SUR-6 regulates nuclear size before mitotic entry, in turn affecting nuclear envelope-based dynein density and motor capacity. Computational simulations predicted the requirement of PP2A-B55/SUR-6 regulation of nuclear size and nuclear-envelope dynein density for proper centrosome separation. Conversely, compromising nuclear lamina integrity led to centrosome detachment from the nuclear envelope and migration defects. Removal of PP2A-B55/SUR-6 and the nuclear lamina simultaneously further disrupted centrosome separation, leading to unseparated centrosome pairs dissociated from the nuclear envelope. Taking these combined results into consideration, we propose a model in which centrosomes migrate and are positioned through the concerted action of PP2A-B55/SUR-6-regulated nuclear envelope-based dynein pulling forces and centrosome-nuclear envelope tethering. Our results add critical precision to models of centrosome separation relative to the nucleus during spindle formation in cell division.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/fisiología , Centrosoma/metabolismo , Proteína Fosfatasa 2/metabolismo , Proteína Fosfatasa 2/fisiología , Animales , Caenorhabditis elegans/metabolismo , Ciclo Celular , Núcleo Celular/metabolismo , Centrosoma/fisiología , Biología Computacional , Simulación por Computador , Dineínas/metabolismo , Mitosis/fisiología , Membrana Nuclear/metabolismo , Lámina Nuclear/metabolismo , Lámina Nuclear/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Huso Acromático/metabolismoRESUMEN
During meiosis, the stepwise release of sister chromatid cohesion is crucial for the equal distribution of genetic material to daughter cells, enabling generation of fertile gametophytes. However, the molecular mechanism that protects centromeric cohesion from release at meiosis I is unclear in Arabidopsis (Arabidopsis thaliana). Here, we report that the protein phosphatase 2A regulatory subunits B'α and B'ß participate in the control of sister chromatid separation. The double mutant b'αß exhibited severe male and female sterility, caused by the lack of a nucleus or presence of an abnormal nucleus in mature microspores and embryo sacs. 4',6-Diamidino-2-phenylindole staining revealed unequal amounts of DNA in the mononuclear microspores. Transverse sections of the anthers revealed unevenly sized tetrads with or without a nucleus, suggesting a defect in meiocyte meiosis. An analysis of chromosome spreads showed that the sister chromatids separated prematurely at anaphase I in b'αß Immunoblotting showed that AtRECOMBINATION DEFECTIVE8 (AtREC8), a key member of the cohesin complex, was hyperphosphorylated in b'αß anthers and pistils during meiosis but hypophosphorylated in the wild type. Furthermore, yeast two-hybrid and bimolecular fluorescence complementation assays showed that B'α and B'ß interact specifically with AtREC8, AtSHUGOSHIN1 (AtSGO1), AtSGO2, and PATRONUS1. Given that B'α was reported to localize to the centromere in meiotic cells, we propose that protein phosphatase 2A B'α and B'ß are recruited by AtSGO1/2 and PATRONUS1 to dephosphorylate AtREC8 at the site of centromere cohesion to shield it from cleavage until anaphase II, contributing to the balanced separation of sister chromatids at meiosis.
Asunto(s)
Proteínas de Arabidopsis/fisiología , Arabidopsis/metabolismo , Centrómero/metabolismo , Meiosis , Proteína Fosfatasa 2/fisiología , Arabidopsis/citología , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromátides/metabolismo , Segregación Cromosómica , Fosforilación , Proteína Fosfatasa 2/genética , Proteína Fosfatasa 2/metabolismo , ReproducciónRESUMEN
Cancer cells depend on a supportive niche (the tumor microenvironment) that promotes tumor cell survival while protecting the malignant cells from therapeutic challenges and the host's defense systems. Cancer cells and the support cells in the tumor microenvironment communicate via cytokines/chemokines, cell:cell contact, or alterations in the metabolic state of the niche (e.g. hypoxia) that promote growth and survival of the tumor cell, influence metastasis, and defeat immune surveillance. These signaling pathways involve dysregulation of not only protein kinases but also protein phosphatases as normal signal transduction processes require both activation and deactivation. For instance, aberrant receptor signaling can result from constitutive activation of a tyrosine kinase such as FLT3 or inactivation of a tyrosine protein phosphatase such as SHP-2 (PTPN11). Activation of serine/threonine kinases such as AKT and ERK are often observed during the development of drug resistance while genomic and non-genomic suppression of serine/threonine protein phosphatases such as PP2A achieve similar results. It is fairly clear that the various protein phosphatases will impact processes that support drug resistance. Of growing interest is the emerging model whereby the support cells in the tumor microenvironment actually serve as drivers of tumorigenesis. This phenomenon has been most prominently observed in osteoblast cells in leukemic niches. At least one protein phosphatase, PTPN11, has emerged as a critical driver of this process in juvenile myelomonocytic leukemia. This review will cover the role of various serine/threonine and tyrosine protein phosphatases in processes that are central to tumor microenvironment function.
Asunto(s)
Fosfoproteínas Fosfatasas/metabolismo , Fosfoproteínas Fosfatasas/fisiología , Microambiente Tumoral/fisiología , Diferenciación Celular/fisiología , Procesos de Crecimiento Celular/fisiología , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Humanos , Leucemia Mieloide/genética , Neoplasias/metabolismo , Neoplasias/fisiopatología , Fosforilación , Proteína Fosfatasa 1/metabolismo , Proteína Fosfatasa 2/metabolismo , Proteína Fosfatasa 2/fisiología , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 11/fisiología , Proteínas Tirosina Quinasas/metabolismo , Transducción de SeñalRESUMEN
Protein Phosphatase 2A (PP2A) encompasses a large family of Ser/Thr phosphatases, consisting of a catalytic C subunit and a structural A subunit that are, in most cases, further bound to a regulatory B-type subunit. The B-type subunits determine function and regulation of PP2A trimers, but despite their importance in PP2A biology, their roles in controlling dephosphorylation of a given substrate in a given cell or tissue remain poorly defined, particularly in the context of a complete organism. Besides twenty PP2A subunit encoding genes, some of which are tissue-specifically expressed, five additional genes encode major regulators of active PP2A trimer assembly, and at least seven genes encode cellular PP2A inhibitors, further adding to the complexity of the mammalian PP2A system. In this review, we summarize current knowledge on physiologic functions of PP2A in germ cell maturation, embryonic development, metabolic regulation, tumor suppression, and homeostasis of adult brain, heart, liver, immune system, lung, intestine, kidney, skin, bone and eye, all retrieved from in vivo studies using PP2A transgenic, knockout or knockin mice. Data from 63 mouse models, generated between 1998 and now, reveal the essentiality of PP2A in vivo, and shed light on tissue-specific functions of particular PP2A subunits on the one hand, and functional redundancies on the other hand. In future, it remains of utmost importance to further characterize the existing models, as well as to generate novel models, with the aim of deepening our insights in PP2A (patho)physiology and, particularly, in the therapeutic potential of PP2A targeting in human disease.
Asunto(s)
Proteína Fosfatasa 2/genética , Proteína Fosfatasa 2/metabolismo , Proteína Fosfatasa 2/fisiología , Animales , Desarrollo Embrionario/fisiología , Genes Supresores de Tumor/fisiología , Células Germinativas/metabolismo , Células Germinativas/fisiología , Holoenzimas/metabolismo , Homeostasis/fisiología , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Metabolismo/fisiología , Ratones , Modelos Animales , Fosfoproteínas Fosfatasas/metabolismo , Fosfoproteínas Fosfatasas/fisiología , Subunidades de Proteína/fisiologíaRESUMEN
Cyclin-dependent kinase (CDK)-activating phosphatases, CDC25A and CDC25B, are labile proteins, and their levels vary in a cell cycle-dependent manner. Immediate-early response IER5 protein negatively regulates the cellular CDC25B levels, and stress-induced IER5 expression potentiates G2/M arrest. IER5 binds to protein phosphatase PP2A and regulates the PP2A substrate specificity. We show that IER5 binds to CDC25B and assists PP2A to convert CDC25B to hypophosphorylated forms. Hypophosphorylation at Ser323 results in the dissociation of CDC25B from 14-3-3 phospho-binding proteins. In IER5 expressing cells, CDC25B dissociated from 14-3-3 is unstable but slightly activated, because 14-3-3 inhibits CDC25B polyubiquitination and CDC25B binding to CDK1. The 14-3-3 binding to CDC25A also impedes CDC25A degradation and CDC25A-CDK2 interaction. We propose that 14-3-3 is an important regulator of CDC25A and CDC25B and that PP2A/IER5 controls the stability and activity of CDC25B through regulating the interaction of CDC25B and 14-3-3.
Asunto(s)
Proteínas 14-3-3/fisiología , Proteína Quinasa CDC2/metabolismo , Proteínas Inmediatas-Precoces/metabolismo , Proteínas Nucleares/metabolismo , Proteína Fosfatasa 2/fisiología , Fosfatasas cdc25/metabolismo , Puntos de Control de la Fase G2 del Ciclo Celular , Células HeLa , Humanos , Puntos de Control de la Fase M del Ciclo CelularRESUMEN
The serine/threonine phosphatase PP2A regulates a vast portion of the phosphoproteome including pathways involved in apoptosis, proliferation and DNA damage response and PP2A inactivation is a vital step in malignant transformation. Many groups have explored the therapeutic venue of combining PP2A reactivation with kinase inhibition to counteract the very changes in tumor suppressors and oncogenes that lead to cancer development. Conversely, inhibition of PP2A to complement chemotherapy and radiation-induced cancer cell death is also an area of active investigation. Here we review the studies that utilize PP2A targeted agents as combination therapy in cancer. A potential role for PP2A in tumor immunity is also highlighted.
Asunto(s)
Proteína Fosfatasa 2/genética , Proteína Fosfatasa 2/metabolismo , Proteína Fosfatasa 2/fisiología , Antineoplásicos/uso terapéutico , Apoptosis/fisiología , Proliferación Celular/fisiología , Reparación del ADN/fisiología , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Terapia Molecular Dirigida/métodos , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Neoplasias/fisiopatología , Neoplasias/terapia , Fosfoproteínas Fosfatasas/metabolismo , Fosfoproteínas Fosfatasas/fisiología , Subunidades de Proteína/fisiología , Transducción de Señal/fisiologíaRESUMEN
Normal functioning of the brain is dependent upon a complex web of communication between numerous cell types. Within neuronal networks, the faithful transmission of information between neurons relies on an equally complex organization of inter- and intra-cellular signaling systems that act to modulate protein activity. In particular, post-translational modifications (PTMs) are responsible for regulating protein activity in response to neurochemical signaling. The key second messenger, cyclic adenosine 3',5'-monophosphate (cAMP), regulates one of the most ubiquitous and influential PTMs, phosphorylation. While cAMP is canonically viewed as regulating the addition of phosphate groups through its activation of cAMP-dependent protein kinases, it plays an equally critical role in regulating removal of phosphate through indirect control of protein phosphatase activity. This dichotomy of regulation by cAMP places it as one of the key regulators of protein activity in response to neuronal signal transduction throughout the brain. In this review we focus on the role of cAMP in regulation of the serine/threonine phosphatases protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A) and the relevance of control of PP1 and PP2A to regulation of brain function and behavior.