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1.
Cancer Res ; 79(16): 4009-4010, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31416848

RESUMO

Critically important to reducing uterine cancer mortality is the development of more effective therapy for aggressive endometrial cancers, including uterine serous cancer and uterine carcinosarcoma, which together account for over half of deaths due to endometrial cancer. About one-third of these aggressive endometrial cancers harbor mutations in the protein phosphatase 2A (PP2A) Aα scaffold subunit encoded by PPP2R1A In this issue, the study by Taylor and colleagues elucidates the role of a highly recurrent PP2A-Aα-subunit mutation PPP2R1A P179R as a biological driver of aggressive endometrial cancer. Compelling data demonstrate that the P179R mutation alters PP2A-Aα protein conformation, impairing holoenzyme formation and reducing PP2A phosphatase activity to promote endometrial cancer progression. Restoration of wild-type PPP2R1A in P179R-mutant endometrial cancer cells increases phosphatase activity and inhibits tumor growth in vivo Furthermore, a small-molecule activator of PP2A (SMAP) phenocopies restoration of wild-type PPP2R1A to suppress tumor growth. These promising results are an important advance toward effective precision therapy for aggressive endometrial cancer.See related article by Taylor et al., p. 4242.


Assuntos
Cistadenocarcinoma Seroso , Neoplasias do Endométrio , Neoplasias Uterinas , Feminino , Humanos , Mutação , Proteína Fosfatase 2/genética
2.
BMC Cancer ; 19(1): 643, 2019 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-31253108

RESUMO

BACKGROUND: Investigations of colorectal carcinogenesis have mainly focused on examining neoplastic tissue. With our aim of identifying potentially cancer-predisposing molecular compositions, we chose a different approach by examining endoscopically normal appearing colonic mucosa of patients with and without colorectal neoplasia (CRN). Directed by this focus, we selected 18 genes that were previously found with altered expression in colorectal cancer affected mucosa. METHODS: Biopsies of colonic mucosa were sampled from 27 patients referred for colonoscopy on suspicion of colorectal disease. Of these, 14 patients had present or previous CRN and the remaining 13 patients served as controls. Using qPCR and Western blot technique, we investigated mRNA and protein expressions. Expressions were investigated for selected kinases in the extracellular signal-regulated kinase/mitogen activated protein kinase (ERK/MAPK), the phosphoinositide 3-kinase/Akt, and the Wnt/ß-catenin pathways as well as for selected phosphatases and several entities associated with prostaglandin E2 (PGE2) signaling. Colonic mucosal contents of PGE2 and PGE2 metabolites were determined by use of ELISA. RESULTS: We found up-regulation of ERK1, ERK2, Akt1, Akt2, PLA2G4A, prostanoid receptor EP3 and phosphatase scaffold subunit PPP2R1B mRNA expression in normal appearing colonic mucosa of CRN patients compared to controls. CONCLUSION: Present study supports that even normal appearing mucosa of CRN patients differs from that of non-CRN patients at a molecular level. Especially expression of ERK1 mRNA was increased (p = 0.007) in CRN group. ERK1 may therefore be considered a potential candidate gene as predictive biomarker for developing CRN. Further validation in larger cohorts are required to determine such predictive use in translational medicine and clinics.


Assuntos
Biomarcadores Tumorais/genética , Neoplasias Colorretais/genética , Predisposição Genética para Doença/genética , Mucosa Intestinal/metabolismo , Biomarcadores Tumorais/metabolismo , Colo/patologia , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Ciclo-Oxigenase 1/metabolismo , Dinoprostona/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Fosfolipases A2 do Grupo IV/genética , Fosfolipases A2 do Grupo IV/metabolismo , Humanos , Hidroxiprostaglandina Desidrogenases/metabolismo , Mucosa Intestinal/patologia , Masculino , Pessoa de Meia-Idade , Proteína Fosfatase 2/genética , Proteína Fosfatase 2/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/metabolismo , Receptores de Prostaglandina E Subtipo EP3/genética , Receptores de Prostaglandina E Subtipo EP3/metabolismo , Transdução de Sinais/genética , Regulação para Cima , beta Catenina/metabolismo
3.
J Vet Med Sci ; 81(7): 1047-1054, 2019 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-31092742

RESUMO

Quercetin is a plant flavonoid that has anti-oxidant, anti-inflammatory, anti-cancer, and anti-ischemic properties. Moreover, quercetin exerts neuroprotective effects against focal cerebral ischemia. Protein phosphatase 2A (PP2A) is a form of serine/threonine phosphatase that modulates various biological functions. Among PP2A subunit types, subunit B exists abundantly in brain tissue and plays an essential function in nervous system. We previously reported the decrease of PP2A subunit B in focal cerebral animal model. This study explored the change of PP2A subunit B expression by quercetin treatment in cerebral ischemic animal model and glutamate-treated hippocampal-derived (HT22) cell culture. Quercetin (10 mg/kg) or vehicle was injected intraperitoneally into male rats before 30 min of middle cerebral artery occlusion (MCAO), and cerebral cortices were isolated 24 hr after MCAO. MCAO induced the neurological behavioral deficit and increased infarct volume. However, quercetin treatment attenuated the increase of neurological deficit and infarction. We detected the alleviation of MCAO-induced the decrease in PP2A subunit B by quercetin treatment using a proteomic approach. Reverse-transcription PCR and Western blot analyses confirmed lower PP2A subunit B expression levels in MCAO group with vehicle. However, quercetin treatment attenuated MCAO-induced this reduction. We also observed the neuroprotective effect of quercetin and the change of PP2A subunit B expression in glutamate-exposed HT22 cells. Glutamate exposure dramatically reduced cell viability and PP2A subunit B expression, and quercetin treatment significantly improved these decreases. We clearly showed that quercetin performs a neuroprotective function and modulates down-regulation of PP2A subunit B against MCAO injury and glutamate toxicity. Thus, our finding suggests that the regulation of PP2A subunit B by quercetin contributes to neuroprotective function in ischemic brain injury.


Assuntos
Isquemia Encefálica/prevenção & controle , Ácido Glutâmico/toxicidade , Fármacos Neuroprotetores/farmacologia , Proteína Fosfatase 2/metabolismo , Quercetina/farmacologia , Animais , Isquemia Encefálica/enzimologia , Isquemia Encefálica/etiologia , Linhagem Celular Transformada , Modelos Animais de Doenças , Infarto da Artéria Cerebral Média/complicações , Infarto da Artéria Cerebral Média/metabolismo , Masculino , Camundongos , Proteína Fosfatase 2/genética , Ratos Sprague-Dawley
4.
Cancer Res ; 79(16): 4242-4257, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31142515

RESUMO

Somatic mutation of the protein phosphatase 2A (PP2A) Aα-subunit gene PPP2R1A is highly prevalent in high-grade endometrial carcinoma. The structural, molecular, and biological basis by which the most recurrent endometrial carcinoma-specific mutation site P179 facilitates features of endometrial carcinoma malignancy has yet to be fully determined. Here, we used a series of structural, biochemical, and biological approaches to investigate the impact of the P179R missense mutation on PP2A function. Enhanced sampling molecular dynamics simulations showed that arginine-to-proline substitution at the P179 residue changes the protein's stable conformation profile. A crystal structure of the tumor-derived PP2A mutant revealed marked changes in A-subunit conformation. Binding to the PP2A catalytic subunit was significantly impaired, disrupting holoenzyme formation and enzymatic activity. Cancer cells were dependent on PP2A disruption for sustained tumorigenic potential, and restoration of wild-type Aα in a patient-derived P179R-mutant cell line restored enzyme function and significantly attenuated tumorigenesis and metastasis in vivo. Furthermore, small molecule-mediated therapeutic reactivation of PP2A significantly inhibited tumorigenicity in vivo. These outcomes implicate PP2A functional inactivation as a critical component of high-grade endometrial carcinoma disease pathogenesis. Moreover, they highlight PP2A reactivation as a potential therapeutic strategy for patients who harbor P179R PPP2R1A mutations. SIGNIFICANCE: This study characterizes a highly recurrent, disease-specific PP2A PPP2R1A mutation as a driver of endometrial carcinoma and a target for novel therapeutic development.See related commentary by Haines and Huang, p. 4009.


Assuntos
Neoplasias do Endométrio , Proteína Fosfatase 2/genética , Carcinogênese , Feminino , Humanos , Mutação , Recidiva Local de Neoplasia
5.
Toxicon ; 166: 76-82, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31121173

RESUMO

Nile tilapia (Oreochromis niloticus) is a freshwater phytoplanktivorous fish species reported to accumulate and tolerate large amounts of cyanotoxins such as microcystins (MCs). The present study aimed to investigate molecular responses to the acute exposure of Nile tilapia to the Microcystin-LA analogue (MC-LA). Thus, the specimens were sublethally exposed to 1000 µg kg-1 of MC-LA for 12, 24, 48, and 96 h. Gene expression of PP1, PP2A, GST, GPX and actin was analyzed by quantitative PCR. The protein abundance profile of PP2A was determined by immunoblotting, while the integrity of its biological function was assessed by a phosphatase enzymatic assay. PP2A activity was significantly and strongly reduced by MC-LA. A resulting feedback mechanism significantly increased PP2A gene expression and protein abundance in all assessed times. However, a recovery of that phosphatase activity was not observed. In this study, the observed increase in GPX gene expression was the only response that could be directly related to the unknown factors associated to the fish survival to such high dose exposure.


Assuntos
Ciclídeos/metabolismo , Microcistinas/toxicidade , Actinas/genética , Actinas/metabolismo , Animais , Ciclídeos/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Glutationa Peroxidase/genética , Glutationa Peroxidase/metabolismo , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Injeções Intraperitoneais , Proteína Fosfatase 1/genética , Proteína Fosfatase 1/metabolismo , Proteína Fosfatase 2/genética , Proteína Fosfatase 2/metabolismo
6.
Eur J Endocrinol ; 180(5): 291-309, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-30893644

RESUMO

Context Most of the knowledge on the factors involved in human sexual development stems from studies of rare cases with disorders of sex development. Here, we have described a novel 46, XY complete gonadal dysgenesis syndrome caused by homozygous variants in PPP2R3C gene. This gene encodes B″gamma regulatory subunit of the protein phosphatase 2A (PP2A), which is a serine/threonine phosphatase involved in the phospho-regulation processes of most mammalian cell types. PPP2R3C gene is most abundantly expressed in testis in humans, while its function was hitherto unknown. Patients and methods Four girls from four unrelated families with 46, XY complete gonadal dysgenesis were studied using exome or Sanger sequencing of PPP2R3C gene. In total, four patients and their heterozygous parents were investigated for clinical, laboratory, immunohistochemical and molecular characteristics. Results We have identified three different homozygous PPP2R3C variants, c.308T>C (p.L103P), c.578T>C (p.L193S) and c.1049T>C (p.F350S), in four girls with 46, XY complete gonadal dysgenesis. Patients also manifested a unique syndrome of extragonadal anomalies, including typical facial gestalt, low birth weight, myopathy, rod and cone dystrophy, anal atresia, omphalocele, sensorineural hearing loss, dry and scaly skin, skeletal abnormalities, renal agenesis and neuromotor delay. We have shown a decreased SOX9-Phospho protein expression in the dysgenetic gonads of the patients with homozygous PPP2R3C variants suggesting impaired SOX9 signaling in the pathogenesis of gonadal dysgenesis. Heterozygous males presented with abnormal sperm morphology and impaired fertility. Conclusion Our findings suggest that PPP2R3C protein is involved in the ontogeny of multiple organs, especially critical for testis development and spermatogenesis. PPPR3C provides insight into pathophysiology, as well as emerging as a potential therapeutic target for male infertility.


Assuntos
Disgenesia Gonadal 46 XY/genética , Proteína Fosfatase 2/genética , Espermatogênese/genética , Adulto , Sequência de Aminoácidos , Sequência de Bases , Criança , Pré-Escolar , Anormalidades Congênitas/genética , Consanguinidade , Feminino , Disgenesia Gonadal 46 XY/patologia , Homozigoto , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Modelos Moleculares , Mutação , Mutação de Sentido Incorreto/genética , Linhagem , Fenótipo , Reação em Cadeia da Polimerase em Tempo Real , Fatores de Transcrição SOX9/genética , Síndrome , Testículo/embriologia , Testículo/patologia
7.
Int J Oncol ; 54(5): 1785-1796, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30864683

RESUMO

Although treatment of chronic myeloid leukemia (CML) has improved with the development of tyrosine kinase inhibitors (TKIs), patients develop fatal blast crisis (BC) whilst receiving TKI treatment. Alternative treatments for cases resistant to TKIs are required. A serine/threonine protein kinase, T­lymphokine­activated killer cell­originated protein kinase (TOPK), is highly expressed in various malignant tumors. Binding of peptides to human leukocyte antigen was assessed via mass spectrometry in K562 CML cells. TOPK expression was assessed in various CML cell lines and in clinical samples obtained from patients with CML using reverse transcription­quantitative polymerase chain reaction and western blot assays. It was observed that TOPK was expressed abundantly in BCR/ABL­positive cell lines and at significantly higher levels in CML clinical samples compared with healthy donor samples. Overexpression of BCR/ABL or the presence of its inhibitor imatinib upregulated and downregulated TOPK expression, respectively, indicating that TOPK may be a target of BCR/ABL. TOPK inhibitor OTS514 suppressed proliferation of BCR/ABL­positive cell lines and colony formation of CD34­positive cells from patients with CML compared with lymphoma patients without bone marrow involvement. Furthermore, phosphorylation of TOPK was increased by protein phosphatase 2A (PP2A) inhibitor okadaic acid and was decreased in the presence of PP2A activator FTY720 compared with untreated samples. As constitutive BCR/ABL activity and inhibition of PP2A are key mechanisms of CML development, TOPK may be a crucial signaling molecule for this disease. Inhibition of TOPK may control disease status of CML, even in cases resistant to TKIs.


Assuntos
Crise Blástica/metabolismo , Proteínas de Fusão bcr-abl/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteína Fosfatase 2/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Crise Blástica/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Proteínas de Fusão bcr-abl/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Antígeno HLA-A24/genética , Antígeno HLA-A24/metabolismo , Humanos , Mesilato de Imatinib/farmacologia , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Masculino , Pessoa de Meia-Idade , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Fosforilação , Proteína Fosfatase 2/genética , Quinolonas/farmacologia , Tiofenos/farmacologia , Adulto Jovem
9.
BMC Pulm Med ; 19(1): 31, 2019 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-30732588

RESUMO

BACKGROUND: Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) has previously been demonstrated to play a pro-inflammatory role in allergic airways disease and COPD through the upregulation of the E3 ubiquitin ligase MID1 and the subsequent deactivation of protein phosphatase 2A (PP2A). METHODS: Biopsies were taken from eight IPF patients presenting to the Second Affiliated Hospital of Jilin University, China between January 2013 and February 2014 with control samples obtained from resected lung cancers. Serum TRAIL, MID1 protein and PP2A activity in biopsies, and patients' lung function were measured. Wild type and TRAIL deficient Tnfsf10-/- BALB/c mice were administered bleomycin to induce fibrosis and some groups were treated with the FTY720 analogue AAL(s) to activate PP2A. Mouse fibroblasts were treated with recombinant TRAIL and fibrotic responses were assessed. RESULTS: TRAIL in serum and MID1 protein levels in biopsies from IPF patients were increased compared to controls. MID1 levels were inversely associated while PP2A activity levels correlated with DLco. Tnfsf10-/- and mice treated with the PP2A activator AAL(s) were largely protected against bleomycin-induced reductions in lung function and fibrotic changes. Addition of recombinant TRAIL to mouse fibroblasts in-vitro increased collagen production which was reversed by PP2A activation with AAL(s). CONCLUSION: TRAIL signalling through MID1 deactivates PP2A and promotes fibrosis with corresponding lung function decline. This may provide novel therapeutic targets for IPF.


Assuntos
Proteínas dos Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Fibrose Pulmonar/patologia , Ligante Indutor de Apoptose Relacionado a TNF/sangue , Fatores de Transcrição/metabolismo , Idoso , Idoso de 80 Anos ou mais , Animais , Estudos de Casos e Controles , China , Colágeno/metabolismo , Feminino , Humanos , Masculino , Camundongos Endogâmicos BALB C , Camundongos Knockout , Proteínas dos Microtúbulos/genética , Pessoa de Meia-Idade , Proteínas Nucleares/genética , Proteína Fosfatase 2/genética , Proteína Fosfatase 2/metabolismo , Proteínas/genética , Proteínas/metabolismo , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/metabolismo , Transdução de Sinais , Ligante Indutor de Apoptose Relacionado a TNF/genética , Fatores de Transcrição/genética , Ubiquitina-Proteína Ligases/metabolismo
10.
J Biol Chem ; 294(15): 5923-5934, 2019 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-30796164

RESUMO

Protein phosphatase 2A (PP2A) represses many oncogenic signaling pathways and is an important tumor suppressor. PP2A comprises three distinct subunits and forms through a highly regulated biogenesis process, with the scaffolding A subunit existing as two highly related isoforms, Aα and Aß. PP2A's tumor-suppressive functions have been intensely studied, and PP2A inactivation has been shown to be a prerequisite for tumor formation. Interestingly, although partial loss of the Aα isoform is growth promoting, complete Aα loss has no transformative properties. Additionally, in cancer patients, Aα is found to be inactivated in a haploinsufficient manner. Using both cellular and in vivo systems, colorectal and endometrial cancer cell lines, and biochemical and cellular assays, here we examined why the complete loss of Aα does not promote tumorigenesis. CRISPR/Cas9-mediated homozygous Aα deletion resulted in decreased colony formation and tumor growth across multiple cell lines. Protein expression analysis of PP2A family members revealed that the Aα deletion markedly up-regulates Aß protein expression by increasing Aß protein stability. Aß knockdown in control and Aα knockout cell lines indicated that Aß is necessary for cell survival in the Aα knockout cells. In the setting of Aα deficiency, co-immunoprecipitation analysis revealed increased binding of specific PP2A regulatory subunits to Aß, and knockdown of these regulatory subunits restored colony-forming ability. Taken together, our results uncover a mechanism by which PP2A Aα regulates Aß protein stability and activity and suggests why homozygous loss of Aα is rarely seen in cancer patients.


Assuntos
Peptídeos beta-Amiloides/biossíntese , Regulação da Expressão Gênica , Proteína Fosfatase 2/metabolismo , Peptídeos beta-Amiloides/genética , Animais , Sistemas CRISPR-Cas , Feminino , Células HCT116 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Ligação Proteica , Proteína Fosfatase 2/genética , Estabilidade Proteica
11.
Plant Physiol ; 179(4): 1556-1568, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30705069

RESUMO

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.


Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/metabolismo , Centrômero/metabolismo , Meiose , Proteína Fosfatase 2/fisiologia , Arabidopsis/citologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromátides/metabolismo , Segregação de Cromossomos , Fosforilação , Proteína Fosfatase 2/genética , Proteína Fosfatase 2/metabolismo , Reprodução
12.
Am J Hum Genet ; 104(1): 139-156, 2019 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-30595372

RESUMO

Type 2A protein phosphatases (PP2As) are highly expressed in the brain and regulate neuronal signaling by catalyzing phospho-Ser/Thr dephosphorylations in diverse substrates. PP2A holoenzymes comprise catalytic C-, scaffolding A-, and regulatory B-type subunits, which determine substrate specificity and physiological function. Interestingly, de novo mutations in genes encoding A- and B-type subunits have recently been implicated in intellectual disability (ID) and developmental delay (DD). We now report 16 individuals with mild to profound ID and DD and a de novo mutation in PPP2CA, encoding the catalytic Cα subunit. Other frequently observed features were severe language delay (71%), hypotonia (69%), epilepsy (63%), and brain abnormalities such as ventriculomegaly and a small corpus callosum (67%). Behavioral problems, including autism spectrum disorders, were reported in 47% of individuals, and three individuals had a congenital heart defect. PPP2CA de novo mutations included a partial gene deletion, a frameshift, three nonsense mutations, a single amino acid duplication, a recurrent mutation, and eight non-recurrent missense mutations. Functional studies showed complete PP2A dysfunction in four individuals with seemingly milder ID, hinting at haploinsufficiency. Ten other individuals showed mutation-specific biochemical distortions, including poor expression, altered binding to the A subunit and specific B-type subunits, and impaired phosphatase activity and C-terminal methylation. Four were suspected to have a dominant-negative mechanism, which correlated with severe ID. Two missense variants affecting the same residue largely behaved as wild-type in our functional assays. Overall, we found that pathogenic PPP2CA variants impair PP2A-B56(δ) functionality, suggesting that PP2A-related neurodevelopmental disorders constitute functionally converging ID syndromes.


Assuntos
Deficiência Intelectual/genética , Mutação , Proteína Fosfatase 2/genética , Adolescente , Criança , Pré-Escolar , Análise Mutacional de DNA , Feminino , Células HEK293 , Haploinsuficiência/genética , Humanos , Masculino , Ligação Proteica/genética , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Síndrome
13.
Biochim Biophys Acta Mol Cell Res ; 1866(1): 31-50, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30030003

RESUMO

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.


Assuntos
Proteína Fosfatase 2/genética , Proteína Fosfatase 2/metabolismo , Proteína Fosfatase 2/fisiologia , Animais , Desenvolvimento Embrionário/fisiologia , Genes Supressores de Tumor/fisiologia , Células Germinativas/metabolismo , Células Germinativas/fisiologia , Holoenzimas/metabolismo , Homeostase/fisiologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Metabolismo/fisiologia , Camundongos , Modelos Animais , Fosfoproteínas Fosfatases/metabolismo , Fosfoproteínas Fosfatases/fisiologia , Subunidades Proteicas/fisiologia
14.
Biochim Biophys Acta Mol Cell Res ; 1866(1): 64-73, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30401536

RESUMO

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.


Assuntos
AMP Cíclico/fisiologia , Proteína Fosfatase 1/fisiologia , Proteína Fosfatase 2/fisiologia , Animais , Encéfalo/metabolismo , Encéfalo/fisiologia , AMP Cíclico/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Fosfoproteína 32 Regulada por cAMP e Dopamina/metabolismo , Inibidores Enzimáticos/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Fosfoproteínas Fosfatases/antagonistas & inibidores , Fosfoproteínas Fosfatases/metabolismo , Fosfoproteínas Fosfatases/fisiologia , Fosforilação , Proteína Fosfatase 1/genética , Proteína Fosfatase 1/metabolismo , Proteína Fosfatase 2/genética , Proteína Fosfatase 2/metabolismo , Transdução de Sinais
15.
J Biol Chem ; 294(7): 2486-2499, 2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30567741

RESUMO

Chronic benzene exposure is associated with hematotoxicity and the development of aplastic anemia and leukemia. However, the signaling pathways underlying benzene-induced hematotoxicity remain to be defined. Here, we investigated the role of protein phosphatase 2A (PP2A) in the regulation of benzene-induced hematotoxicity in a murine model. Male mice with a hepatocyte-specific homozygous deletion of the Ppp2r1a gene (encoding PP2A Aα subunit) (HO) and matched wildtype (WT) mice were exposed to benzene via inhalation at doses of 1, 10, and 100 ppm for 28 days. Peripheral white blood cell counts and activation of bone marrow progenitors were attenuated in the HO mice, indicating that Ppp2r1a deletion protects against benzene-induced hematotoxicity. Moreover, elevation of urinary S-phenyl mercapturic acid, a benzene metabolite, was much greater in WT mice than in HO mice. Real-time exhalation analysis revealed more exhaled benzene but fewer benzene metabolites in HO mice than in WT mice, possibly because of the down-regulation of Cyp2e1, encoding cytochrome P4502E1, in hepatocytes of the HO mice. Loss-of-function screening disclosed that PP2A complexes containing the B56α subunit participate in regulating Cyp2e1 expression. Notably, PP2A-B56α suppression in HepG2 cells resulted in persistent ß-catenin phosphorylation at Ser33-Ser37-Thr41 in response to CYP2E1 agonists. In parallel, nuclear translocation of ß-catenin was inhibited, concomitant with a remarkable decrease of Cyp2e1 expression. These findings support the notion that a regulatory cascade comprising PP2A-B56α, ß-catenin, and Cyp2e1 is involved in benzene-induced hematotoxicity, providing critical insight into the role of PP2A in responses to the environmental chemicals.


Assuntos
Benzeno/toxicidade , Citocromo P-450 CYP2E1/biossíntese , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Proteína Fosfatase 2/metabolismo , Transcrição Genética/efeitos dos fármacos , Animais , Citocromo P-450 CYP2E1/genética , Células Hep G2 , Humanos , Camundongos , Camundongos Knockout , Proteína Fosfatase 2/genética
16.
J Agric Food Chem ; 67(1): 441-451, 2019 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-30562020

RESUMO

γ-Tocotrienol (γ-T3) exhibits the activity of anticancer via regulating cell signaling pathways. Nuclear factor-κB (NF-κB), one of the crucial pro-inflammatory factors, is involved in the regulation of cell proliferation, apoptosis, invasion, and migration of tumor. In the present study, NF-κB activity inhibited by γ-T3 was investigated in gastric cancer cells. Cell proliferation, NF-κB activity, active protein phosphatase type 2A (PP2A), and ataxia-telangiectasia mutated (ATM) protein were explored using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT), methylene blue, enzyme-linked immunosorbent assay (ELISA), malachite green, luciferase, and Western blotting assays. The effects of γ-T3 on tumor growth and the expression of NF-κB and PP2A proteins were also further examined by implanting human gastric cancer cells in a BALB/c nude mouse model. The results showed that γ-T3 significantly inhibited the cell proliferation and attenuated the NF-κB activity in vitro and in vivo. γ-T3 dramatically increased PP2A activity and protein expression, which suppressed ATM phosphorylation and its translocation to the cytoplasm in gastric cancer cells. Thus, our findings may provide mechanistic insight into effects of γ-T3 on the regulation of NF-κB activity by a PP2A-dependent mechanism and suggest that PP2A may serve as a molecular target for a potential chemopreventive agent.


Assuntos
Proliferação de Células/efeitos dos fármacos , Cromanos/administração & dosagem , NF-kappa B/metabolismo , Neoplasias Gástricas/tratamento farmacológico , Vitamina E/análogos & derivados , Animais , Apoptose/efeitos dos fármacos , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , NF-kappa B/genética , Proteína Fosfatase 2/genética , Proteína Fosfatase 2/metabolismo , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/fisiopatologia , Vitamina E/administração & dosagem
17.
Genes Cells ; 24(2): 172-186, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30584685

RESUMO

Cell proliferation and cellular quiescence/G0 phase must be regulated in response to intra-/extracellular environments, and such regulation is achieved by the orchestration of protein kinases and protein phosphatases. Here, we investigated fission yeast potential orthologs (Cek1, Ppk18 and Ppk31) of the metazoan Greatwall kinase (Gwl), which inhibits type-2A protein phosphatase with B55 subunit (PP2AB55 ) by phosphorylating and activating the PP2AB55 inhibitors, α-endosulfine/ARPP-19 (Ensa/ARPP-19). Gwl and Ensa/ARPP-19 regulate mitosis; however, we found Ppk18, Cek1 and Mug134/Igo1, the counterpart of Ensa/ARPP-19, are not essential for normal mitosis but regulate nitrogen starvation (-N)-induced proper G0 entry and maintenance. Genetic and biochemical analyses indicated that the conserved Gwl site (serine 64) was phosphorylated in the G0 phase in a Ppk18-dependent manner, and the phosphorylated Mug134/Igo1 inhibited PP2AB55 in vitro. The alanine substitution of the serine 64 caused defects in G0 entry and maintenance as well as the mug134/igo1+ deletion. These results indicate that PP2AB55 activity must be regulated properly to establish the G0 phase. Consistently, simultaneous deletion of the B55 gene with mug134/igo1+ partially rescued the Mug134/Igo1 mutant phenotype. We suggest that in fission yeast, PP2AB55 regulation by the Ppk18-Mug134/Igo1 pathway is required for G0 entry and establishment of robust viability during the G0 phase.


Assuntos
Mitose , Peptídeos/metabolismo , Fase de Repouso do Ciclo Celular , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Transdução de Sinais , Sequência de Aminoácidos , Fosforilação , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteína Fosfatase 2/antagonistas & inibidores , Proteína Fosfatase 2/genética , Proteína Fosfatase 2/metabolismo , Schizosaccharomyces/citologia , Schizosaccharomyces/genética , Schizosaccharomyces/crescimento & desenvolvimento , Proteínas de Schizosaccharomyces pombe/genética , Homologia de Sequência
18.
J Biol Chem ; 294(10): 3419-3431, 2019 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-30587577

RESUMO

Several protein kinases, including protein kinase C, Ca2+/calmodulin-dependent protein kinase II, and extracellular signal-regulated kinase, play key roles in the regulation of dopamine transporter (DAT) functions. These functions include surface expression, internalization, and forward and reverse transport, with phosphorylation sites for these kinases being linked to distinct regions of the DAT N terminus. Protein phosphatases (PPs) also regulate DAT activity, but the specific residues associated with their activities have not yet been elucidated. In this study, using co-immunoprecipitation followed by MS and immunoblotting analyses, we demonstrate the association of DAT with PP1 and PP2A in the mouse brain and heterologous cell systems. By applying MS in conjunction with a metabolic labeling method, we defined a PP1/2A-sensitive phosphorylation site at Thr-48 in human DAT, a residue that has not been previously reported to be involved in DAT phosphorylation. Site-directed mutagenesis of Thr-48 to Ala (T48A) to prevent phosphorylation enhanced dopamine transport kinetics, supporting a role for this residue in regulating DAT activity. Moreover, T48A-DAT displayed increased palmitoylation, suggesting that phosphorylation/dephosphorylation at this site has an additional regulatory role and reinforcing a previously reported reciprocal relationship between C-terminal palmitoylation and N-terminal phosphorylation.


Assuntos
Encéfalo/metabolismo , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Dopamina/metabolismo , Proteína Fosfatase 1/metabolismo , Proteína Fosfatase 2/metabolismo , Animais , Transporte Biológico Ativo/fisiologia , Dopamina/genética , Proteínas da Membrana Plasmática de Transporte de Dopamina/genética , Humanos , Lipoilação/genética , Camundongos , Camundongos Knockout , Fosforilação , Proteína Fosfatase 1/genética , Proteína Fosfatase 2/genética , Treonina/genética , Treonina/metabolismo
19.
Biochim Biophys Acta Mol Cell Res ; 1866(1): 51-63, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30401535

RESUMO

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.


Assuntos
Proteína Fosfatase 2/genética , Proteína Fosfatase 2/metabolismo , Proteína Fosfatase 2/fisiologia , Antineoplásicos/uso terapêutico , Apoptose/fisiologia , Proliferação de Células/fisiologia , Reparo do DNA/fisiologia , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Terapia de Alvo Molecular/métodos , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Neoplasias/fisiopatologia , Neoplasias/terapia , Fosfoproteínas Fosfatases/metabolismo , Fosfoproteínas Fosfatases/fisiologia , Subunidades Proteicas/fisiologia , Transdução de Sinais/fisiologia
20.
Am J Pathol ; 189(1): 132-146, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30553437

RESUMO

Cartilage oligomeric matrix protein (COMP) is a large, multifunctional extracellular protein that, when mutated, is retained in the rough endoplasmic reticulum (ER). This retention elicits ER stress, inflammation, and oxidative stress, resulting in dysfunction and death of growth plate chondrocytes. While identifying the cellular pathologic mechanisms underlying the murine mutant (MT)-COMP model of pseudoachondroplasia, increased midline-1 (MID1) expression and mammalian target of rapamycin complex 1 (mTORC1) signaling was found. This novel role for MID1/mTORC1 signaling was investigated since treatments shown to repress the pathology also reduced Mid1/mTORC1. Although ER stress-inducing drugs or tumor necrosis factor α (TNFα) in rat chondrosarcoma cells increased Mid1, oxidative stress did not, establishing that ER stress- or TNFα-driven inflammation alone is sufficient to elevate MID1 expression. Since MID1 ubiquitinates protein phosphatase 2A (PP2A), a negative regulator of mTORC1, PP2A was evaluated in MT-COMP growth plate chondrocytes. PP2A was decreased, indicating de-repression of mTORC1 signaling. Rapamycin treatment in MT-COMP mice reduced mTORC1 signaling and intracellular retention of COMP, and increased proliferation, but did not change inflammatory markers IL-16 and eosinophil peroxidase. Lastly, mRNA from tuberous sclerosis-1/2-null mice brain tissue exhibiting ER stress had increased Mid1 expression, confirming the relationship between ER stress and MID1/mTORC1 signaling. These findings suggest a mechanistic link between ER stress and MID1/mTORC1 signaling that has implications extending to other conditions involving ER stress.


Assuntos
Acondroplasia , Proteína de Matriz Oligomérica de Cartilagem , Sistemas de Liberação de Medicamentos , Alvo Mecanístico do Complexo 1 de Rapamicina , Acondroplasia/tratamento farmacológico , Acondroplasia/genética , Acondroplasia/patologia , Animais , Biomarcadores/metabolismo , Proteína de Matriz Oligomérica de Cartilagem/genética , Proteína de Matriz Oligomérica de Cartilagem/metabolismo , Linhagem Celular Tumoral , Condrócitos/metabolismo , Condrócitos/patologia , Modelos Animais de Doenças , Estresse do Retículo Endoplasmático/genética , Retículo Endoplasmático Rugoso/genética , Retículo Endoplasmático Rugoso/metabolismo , Retículo Endoplasmático Rugoso/patologia , Peroxidase de Eosinófilo/genética , Peroxidase de Eosinófilo/metabolismo , Humanos , Mediadores da Inflamação/metabolismo , Interleucina-16/genética , Interleucina-16/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Camundongos Transgênicos , Mutação/genética , Proteína Fosfatase 2/genética , Proteína Fosfatase 2/metabolismo , Proteínas/genética , Proteínas/metabolismo , Ratos , Transdução de Sinais/genética , Sirolimo/farmacologia , Proteína 1 do Complexo Esclerose Tuberosa/genética , Proteína 1 do Complexo Esclerose Tuberosa/metabolismo , Proteína 2 do Complexo Esclerose Tuberosa/genética , Proteína 2 do Complexo Esclerose Tuberosa/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo
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