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1.
Cancer Cell ; 35(5): 798-815.e5, 2019 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-31031016

RESUMO

Tumor cells may adapt to metabolic challenges by alternating between glycolysis and oxidative phosphorylation (OXPHOS). To target this metabolic plasticity, we combined intermittent fasting, a clinically feasible approach to reduce glucose availability, with the OXPHOS inhibitor metformin. In mice exposed to 24-h feeding/fasting cycles, metformin impaired tumor growth only when administered during fasting-induced hypoglycemia. Synergistic anti-neoplastic effects of the metformin/hypoglycemia combination were mediated by glycogen synthase kinase 3ß (GSK3ß) activation downstream of PP2A, leading to a decline in the pro-survival protein MCL-1, and cell death. Mechanistically, specific activation of the PP2A-GSK3ß axis was the sum of metformin-induced inhibition of CIP2A, a PP2A suppressor, and of upregulation of the PP2A regulatory subunit B56δ by low glucose, leading to an active PP2A-B56δ complex with high affinity toward GSK3ß.

3.
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
4.
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
5.
Int J Mol Sci ; 19(8)2018 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-30104481

RESUMO

Type II endometrial carcinomas (ECs) are responsible for most endometrial cancer-related deaths due to their aggressive nature, late stage detection and high tolerance for standard therapies. However, there are no targeted therapies for type II ECs, and they are still treated the same way as the clinically indolent and easily treatable type I ECs. Therefore, type II ECs are in need of new treatment options. More recently, molecular analysis of endometrial cancer revealed phosphorylation-dependent oncogenic signalling in the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways to be most frequently altered in type II ECs. Consequently, clinical trials tested pharmacologic kinase inhibitors targeting these pathways, although mostly with rather disappointing results. In this review, we highlight the most common genetic alterations in type II ECs. Additionally, we reason why most clinical trials for ECs using targeted kinase inhibitors had unsatisfying results and what should be changed in future clinical trial setups. Furthermore, we argue that, besides kinases, phosphatases should no longer be ignored in clinical trials, particularly in type II ECs, where the tumour suppressive phosphatase protein phosphatase type 2A (PP2A) is frequently mutated. Lastly, we discuss the therapeutic potential of targeting PP2A for (re)activation, possibly in combination with pharmacologic kinase inhibitors.


Assuntos
Neoplasias do Endométrio/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Neoplasias do Endométrio/genética , Neoplasias do Endométrio/patologia , Feminino , Humanos , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosfatidilinositol 3-Quinases/antagonistas & inibidores , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteína Fosfatase 2/antagonistas & inibidores , Proteína Fosfatase 2/metabolismo , Transdução de Sinais/efeitos dos fármacos
6.
FEBS Lett ; 592(14): 2432-2443, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29933512

RESUMO

The protein kinase D (PKD) family is regulated through multi-site phosphorylation, including autophosphorylation. For example, PKD displays in vivo autophosphorylation on Ser-742 (and Ser-738 in vitro) in the activation loop and Ser-910 in the C-tail (hPKD1 numbering). In this paper, we describe the surprising observation that PKD also displays in vitro autocatalytic activity towards a Tyr residue in the P + 1 loop of the activation segment. We define the molecular determinants for this unusual activity and identify a Cys residue (C705 in PKD1) in the catalytic loop as of utmost importance. In cells, PKD Tyr autophosphorylation is suppressed through the association of an inhibitory factor. Our findings provide important novel insights into PKD (auto)regulation.

7.
Nat Commun ; 9(1): 2227, 2018 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-29884836

RESUMO

SAMHD1 is a critical restriction factor for HIV-1 in non-cycling cells and its antiviral activity is regulated by T592 phosphorylation. Here, we show that SAMHD1 dephosphorylation at T592 is controlled during the cell cycle, occurring during M/G1 transition in proliferating cells. Using several complementary proteomics and biochemical approaches, we identify the phosphatase PP2A-B55α responsible for rendering SAMHD1 antivirally active. SAMHD1 is specifically targeted by PP2A-B55α holoenzymes during mitotic exit, in line with observations that PP2A-B55α is a key mitotic exit phosphatase in mammalian cells. Strikingly, as HeLa or activated primary CD4+ T cells enter the G1 phase, pronounced reduction of RT products is observed upon HIV-1 infection dependent on the presence of dephosphorylated SAMHD1. Moreover, PP2A controls SAMHD1 pT592 level in non-cycling monocyte-derived macrophages (MDMs). Thus, the PP2A-B55α holoenzyme is a key regulator to switch on the antiviral activity of SAMHD1.

8.
Int J Biochem Cell Biol ; 96: 98-134, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29031806

RESUMO

Aberrant protein phosphorylation is one of the hallmarks of cancer cells, and in many cases a prerequisite to sustain tumor development and progression. Like protein kinases, protein phosphatases are key regulators of cell signaling. However, their contribution to aberrant signaling in cancer cells is overall less well appreciated, and therefore, their clinical potential remains largely unexploited. In this review, we provide an overview of tumor suppressive protein phosphatases in human cancer. Along their mechanisms of inactivation in defined cancer contexts, we give an overview of their functional roles in diverse signaling pathways that contribute to their tumor suppressive abilities. Finally, we discuss their emerging roles as predictive or prognostic markers, their potential as synthetic lethality targets, and the current feasibility of their reactivation with pharmacologic compounds as promising new cancer therapies. We conclude that their inclusion in clinical practice has obvious potential to significantly improve therapeutic outcome in various ways, and should now definitely be pushed forward.

9.
Cancer Res ; 77(24): 6825-6837, 2017 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-29046336

RESUMO

Protein phosphatase 2A (PP2A) complexes counteract many oncogenic kinase pathways. In cancer cells, PP2A function can be compromised by several mechanisms, including sporadic mutations in its scaffolding A and regulatory B subunits or more frequently through overexpression of cellular PP2A inhibitors. Here, we identify a novel genetic mechanism by which PP2A function is recurrently affected in human cancer, involving haploinsufficiency of PPP2R4, a gene encoding the cellular PP2A activator PTPA. Notably, up to 70% of cancer patients showed a heterozygous deletion or missense mutations in PPP2R4 Cancer-associated PTPA mutants exhibited decreased abilities to bind the PP2A-C subunit or activate PP2A and failed to reverse the tumorigenic phenotype induced by PTPA suppression, indicating they function as null alleles. In Ppp2r4 gene-trapped (gt) mice showing residual PTPA expression, total PP2A activity and methylation were reduced, selectively affecting specific PP2A holoenzymes. Both PTPAgt/gt and PTPA+/gt mice showed higher rates of spontaneous tumors, mainly hematologic malignancies and hepatocellular adenomas and carcinomas. These tumors exhibited increased c-Myc phosphorylation and increased Wnt or Hedgehog signaling. We observed a significant reduction in lifespan in PTPA+/gt mice compared with wild-type mice. In addition, chemical-induced skin carcinogenesis was accelerated in PTPA+/gt compared with wild-type mice. Our results provide evidence for PPP2R4 as a haploinsufficient tumor suppressor gene, defining a high-penetrance genetic mechanism for PP2A inhibition in human cancer. Cancer Res; 77(24); 6825-37. ©2017 AACR.


Assuntos
Haploinsuficiência , Neoplasias/genética , Fosfoproteínas Fosfatases/genética , Proteína Fosfatase 2/metabolismo , Animais , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Células Cultivadas , Ativação Enzimática/genética , Feminino , Genes Supressores de Tumor/fisiologia , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neoplasias/metabolismo
10.
Nat Commun ; 8(1): 922, 2017 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-29030545

RESUMO

Yeast and cancer cells share the unusual characteristic of favoring fermentation of sugar over respiration. We now reveal an evolutionary conserved mechanism linking fermentation to activation of Ras, a major regulator of cell proliferation in yeast and mammalian cells, and prime proto-oncogene product. A yeast mutant (tps1∆) with overactive influx of glucose into glycolysis and hyperaccumulation of Fru1,6bisP, shows hyperactivation of Ras, which causes its glucose growth defect by triggering apoptosis. Fru1,6bisP is a potent activator of Ras in permeabilized yeast cells, likely acting through Cdc25. As in yeast, glucose triggers activation of Ras and its downstream targets MEK and ERK in mammalian cells. Biolayer interferometry measurements show that physiological concentrations of Fru1,6bisP stimulate dissociation of the pure Sos1/H-Ras complex. Thermal shift assay confirms direct binding to Sos1, the mammalian ortholog of Cdc25. Our results suggest that the Warburg effect creates a vicious cycle through Fru1,6bisP activation of Ras, by which enhanced fermentation stimulates oncogenic potency.Yeast and cancer cells both favor sugar fermentation in aerobic conditions. Here the authors describe a conserved mechanism from yeast to mammals where the glycolysis intermediate fructose-1,6-bisphosphate binds Cdc25/Sos1 and couples increased glycolytic flux to increased Ras proto-oncoprotein activity.


Assuntos
Frutosefosfatos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas ras/metabolismo , Animais , Fermentação , Glucose/metabolismo , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Glicólise , Proteína SOS1/genética , Proteína SOS1/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Proteínas ras/genética , ras-GRF1/genética , ras-GRF1/metabolismo
11.
Sci Rep ; 7(1): 887, 2017 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-28428613

RESUMO

Protein kinases are essential molecules in life and their crucial function requires tight regulation. Many kinases are regulated via phosphorylation within their activation loop. This loop is embedded in the activation segment, which additionally contains the Mg2+ binding loop and a P + 1 loop that is important in substrate binding. In this report, we identify Abl-mediated phosphorylation of a highly conserved Tyr residue in the P + 1 loop of protein kinase D2 (PKD2) during oxidative stress. Remarkably, we observed that the three human PKD isoforms display very different degrees of P + 1 loop Tyr phosphorylation and we identify one of the molecular determinants for this divergence. This is paralleled by a different activation mechanism of PKD1 and PKD2 during oxidative stress. Tyr phosphorylation in the P + 1 loop of PKD2 increases turnover for Syntide-2, while substrate specificity and the role of PKD2 in NF-κB signaling remain unaffected. Importantly, Tyr to Phe substitution renders the kinase inactive, jeopardizing its use as a non-phosphorylatable mutant. Since large-scale proteomics studies identified P + 1 loop Tyr phosphorylation in more than 70 Ser/Thr kinases in multiple conditions, our results do not only demonstrate differential regulation/function of PKD isoforms under oxidative stress, but also have implications for kinase regulation in general.


Assuntos
Estresse Oxidativo , Proteína Quinase C/metabolismo , Processamento de Proteína Pós-Traducional , Sequência Conservada , Células HEK293 , Células HeLa , Humanos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , NF-kappa B/metabolismo , Peptídeos/metabolismo , Fosforilação , Domínios Proteicos , Proteína Quinase C/química , Proteína Quinase C/genética , Tirosina/genética , Tirosina/metabolismo
12.
Oncoscience ; 4(11-12): 154-155, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-29344545
13.
Cancer Res ; 76(19): 5719-5731, 2016 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-27485451

RESUMO

Somatic missense mutations in the Ser/Thr protein phosphatase 2A (PP2A) Aα scaffold subunit gene PPP2R1A are among the few genomic alterations that occur frequently in serous endometrial carcinoma (EC) and carcinosarcoma, two clinically aggressive subtypes of uterine cancer with few therapeutic options. Previous studies reported that cancer-associated Aα mutants exhibit defects in binding to other PP2A subunits and contribute to cancer development by a mechanism of haploinsufficiency. Here we report on the functional significance of the most recurrent PPP2R1A mutations in human EC, which cluster in Aα HEAT repeats 5 and 7. Beyond predicted loss-of-function effects on the formation of a subset of PP2A holoenzymes, we discovered that Aα mutants behave in a dominant-negative manner due to gain-of-function interactions with the PP2A inhibitor TIPRL1. Dominant-negative Aα mutants retain binding to specific subunits of the B56/B' family and form substrate trapping complexes with impaired phosphatase activity via increased recruitment of TIPRL1. Accordingly, overexpression of the Aα mutants in EC cells harboring wild-type PPP2R1A increased anchorage-independent growth and tumor formation, and triggered hyperphosphorylation of oncogenic PP2A-B56/B' substrates in the GSK3ß, Akt, and mTOR/p70S6K signaling pathways. TIPRL1 silencing restored GSK3ß phosphorylation and rescued the EC cell growth advantage. Our results reveal how PPP2R1A mutations affect PP2A function and oncogenic signaling, illuminating the genetic basis for serous EC development and its potential control by rationally targeted therapies. Cancer Res; 76(19); 5719-31. ©2016 AACR.


Assuntos
Cistadenocarcinoma Seroso/genética , Neoplasias do Endométrio/genética , Mutação de Sentido Incorreto , Proteína Fosfatase 2/genética , Animais , Linhagem Celular Tumoral , Proliferação de Células , Cistadenocarcinoma Seroso/etiologia , Cistadenocarcinoma Seroso/patologia , Neoplasias do Endométrio/etiologia , Neoplasias do Endométrio/patologia , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Camundongos , Proteínas Quinases S6 Ribossômicas 70-kDa/fisiologia , Serina-Treonina Quinases TOR/fisiologia
14.
J Cell Sci ; 129(8): 1605-18, 2016 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-26945059

RESUMO

Here, we identify the LIM protein lipoma-preferred partner (LPP) as a binding partner of a specific protein phosphatase 2A (PP2A) heterotrimer that is characterised by the regulatory PR130/B″α1 subunit (encoded by PPP2R3A). The PR130 subunit interacts with the LIM domains of LPP through a conserved Zn²âº-finger-like motif in the differentially spliced N-terminus of PR130. Isolated LPP-associated PP2A complexes are catalytically active. PR130 colocalises with LPP at multiple locations within cells, including focal contacts, but is specifically excluded from mature focal adhesions, where LPP is still present. An LPP-PR130 fusion protein only localises to focal adhesions upon deletion of the domain of PR130 that binds to the PP2A catalytic subunit (PP2A/C), suggesting that PR130-LPP complex formation is dynamic and that permanent recruitment of PP2A activity might be unfavourable for focal adhesion maturation. Accordingly, siRNA-mediated knockdown of PR130 increases adhesion of HT1080 fibrosarcoma cells onto collagen I and decreases their migration in scratch wound and Transwell assays. Complex formation with LPP is mandatory for these PR130-PP2A functions, as neither phenotype can be rescued by re-expression of a PR130 mutant that no longer binds to LPP. Our data highlight the importance of specific, locally recruited PP2A complexes in cell adhesion and migration dynamics.


Assuntos
Proteínas do Citoesqueleto/metabolismo , Proteínas com Domínio LIM/metabolismo , Proteína Fosfatase 2/metabolismo , Domínio Catalítico/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Adesões Focais/genética , Humanos , Ligação Proteica , Proteína Fosfatase 2/genética , RNA Interferente Pequeno/genética
15.
J Cell Sci ; 129(6): 1101-14, 2016 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-26826186

RESUMO

Phosphoinositides, particularly phosphatidylinositol (3,4,5)-trisphosphate [PI(3,4,5)P3] and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2], are recognized by SHIP2 (also known as INPPL1) a member of the inositol polyphosphate 5-phosphatase family. SHIP2 dephosphorylates PI(3,4,5)P3 to form PI(3,4)P2; the latter interacts with specific target proteins (e.g. lamellipodin). Although the preferred SHIP2 substrate is PI(3,4,5)P3, PI(4,5)P2 can also be dephosphorylated by this enzyme to phosphatidylinositol 4-phosphate (PI4P). Through depletion of SHIP2 in the glioblastoma cell line 1321 N1, we show that SHIP2 inhibits cell migration. In different glioblastoma cell lines and primary cultures, SHIP2 staining at the plasma membrane partly overlaps with PI(4,5)P2 immunoreactivity. PI(4,5)P2 was upregulated in SHIP2-deficient N1 cells as compared to control cells; in contrast, PI4P was very much decreased in SHIP2-deficient cells. Therefore, SHIP2 controls both PI(3,4,5)P3 and PI(4,5)P2 levels in intact cells. In 1321 N1 cells, the PI(4,5)P2-binding protein myosin-1c was identified as a new interactor of SHIP2. Regulation of PI(4,5)P2 and PI4P content by SHIP2 controls 1321 N1 cell migration through the organization of focal adhesions. Thus, our results reveal a new role of SHIP2 in the control of PI(4,5)P2, PI4P and cell migration in PTEN-deficient glioblastoma 1321 N1 cells.


Assuntos
Membrana Celular/metabolismo , Movimento Celular , Glioblastoma/enzimologia , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/metabolismo , Linhagem Celular Tumoral , Membrana Celular/genética , Adesões Focais/genética , Adesões Focais/metabolismo , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/fisiopatologia , Humanos , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/genética
16.
J Clin Invest ; 125(8): 3051-62, 2015 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-26168268

RESUMO

Here we report inherited dysregulation of protein phosphatase activity as a cause of intellectual disability (ID). De novo missense mutations in 2 subunits of serine/threonine (Ser/Thr) protein phosphatase 2A (PP2A) were identified in 16 individuals with mild to severe ID, long-lasting hypotonia, epileptic susceptibility, frontal bossing, mild hypertelorism, and downslanting palpebral fissures. PP2A comprises catalytic (C), scaffolding (A), and regulatory (B) subunits that determine subcellular anchoring, substrate specificity, and physiological function. Ten patients had mutations within a highly conserved acidic loop of the PPP2R5D-encoded B56δ regulatory subunit, with the same E198K mutation present in 6 individuals. Five patients had mutations in the PPP2R1A-encoded scaffolding Aα subunit, with the same R182W mutation in 3 individuals. Some Aα cases presented with large ventricles, causing macrocephaly and hydrocephalus suspicion, and all cases exhibited partial or complete corpus callosum agenesis. Functional evaluation revealed that mutant A and B subunits were stable and uncoupled from phosphatase activity. Mutant B56δ was A and C binding-deficient, while mutant Aα subunits bound B56δ well but were unable to bind C or bound a catalytically impaired C, suggesting a dominant-negative effect where mutant subunits hinder dephosphorylation of B56δ-anchored substrates. Moreover, mutant subunit overexpression resulted in hyperphosphorylation of GSK3ß, a B56δ-regulated substrate. This effect was in line with clinical observations, supporting a correlation between the ID degree and biochemical disturbance.


Assuntos
Agenesia do Corpo Caloso , Corpo Caloso , Transtornos Mentais , Mutação de Sentido Incorreto , Proteína Fosfatase 2 , Adolescente , Adulto , Agenesia do Corpo Caloso/enzimologia , Agenesia do Corpo Caloso/genética , Agenesia do Corpo Caloso/patologia , Substituição de Aminoácidos , Criança , Pré-Escolar , Corpo Caloso/enzimologia , Corpo Caloso/patologia , Feminino , Quinase 3 da Glicogênio Sintase/genética , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Humanos , Lactente , Masculino , Transtornos Mentais/enzimologia , Transtornos Mentais/genética , Transtornos Mentais/patologia , Pessoa de Meia-Idade , Fosforilação/genética , Proteína Fosfatase 2/genética , Proteína Fosfatase 2/metabolismo , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína
17.
FEBS Lett ; 588(5): 727-32, 2014 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-24462681

RESUMO

Deoxycytidine kinase (dCK) is a critical enzyme for activation of anticancer nucleoside analogs. Its activity is controlled via Ser-74 phosphorylation. Here, we investigated which Ser/Thr phosphatase dephosphorylates Ser-74. In cells, the PP1/PP2A inhibitor okadaic acid increased both dCK activity and Ser-74 phosphorylation at concentrations reported to specifically target PP2A. In line with this, purified PP2A, but not PP1, dephosphorylated recombinant pSer-74-dCK. In cell lysates, the Ser-74-dCK phosphatase activity was found to be latent, Mn(2+)-activated, responsive to PP2A inhibitors, and diminished after PP2A-immunodepletion. Use of siRNAs allowed concluding definitively that PP2A constitutively dephosphorylates dCK in cells and negatively regulates its activity.


Assuntos
Desoxicitidina Quinase/metabolismo , Fosfoproteínas Fosfatases/fisiologia , Processamento de Proteína Pós-Traducional , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Linhagem Celular Tumoral , Células HEK293 , Humanos , Ácido Okadáico/farmacologia , Fosfoproteínas Fosfatases/antagonistas & inibidores , Fosforilação , Proteína Fosfatase 2C , Serina/metabolismo
18.
Front Oncol ; 4: 347, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25566494

RESUMO

Reversible protein phosphorylation plays a crucial role in regulating cell signaling. In normal cells, phosphoregulation is tightly controlled by a network of protein kinases counterbalanced by several protein phosphatases. Deregulation of this delicate balance is widely recognized as a central mechanism by which cells escape external and internal self-limiting signals, eventually resulting in malignant transformation. A large fraction of hematologic malignancies is characterized by constitutive or unrestrained activation of oncogenic kinases. This is in part achieved by activating mutations, chromosomal rearrangements, or constitutive activation of upstream kinase regulators, in part by inactivation of their anti-oncogenic phosphatase counterparts. Protein phosphatase 2A (PP2A) represents a large family of cellular serine/threonine phosphatases with suspected tumor suppressive functions. In this review, we highlight our current knowledge about the complex structure and biology of these phosphatases in hematologic cells, thereby providing the rationale behind their diverse signaling functions. Eventually, this basic knowledge is a key to truly understand the tumor suppressive role of PP2A in leukemogenesis and to allow further rational development of therapeutic strategies targeting PP2A.

19.
EMBO J ; 32(18): 2491-503, 2013 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-23955003

RESUMO

To supply tissues with nutrients and oxygen, the cardiovascular system forms a seamless, hierarchically branched, network of lumenized tubes. Here, we show that maintenance of patent vessel lumens requires the Bα regulatory subunit of protein phosphatase 2A (PP2A). Deficiency of Bα in zebrafish precludes vascular lumen stabilization resulting in perfusion defects. Similarly, inactivation of PP2A-Bα in cultured ECs induces tubulogenesis failure due to alteration of cytoskeleton dynamics, actomyosin contractility and maturation of cell-extracellular matrix (ECM) contacts. Mechanistically, we show that PP2A-Bα controls the activity of HDAC7, an essential transcriptional regulator of vascular stability. In the absence of PP2A-Bα, transcriptional repression by HDAC7 is abrogated leading to enhanced expression of the cytoskeleton adaptor protein ArgBP2. ArgBP2 hyperactivates RhoA causing inadequate rearrangements of the EC actomyosin cytoskeleton. This study unravels the first specific role for a PP2A holoenzyme in development: the PP2A-Bα/HDAC7/ArgBP2 axis maintains vascular lumens by balancing endothelial cytoskeletal dynamics and cell-matrix adhesion.


Assuntos
Endotélio Vascular/fisiologia , Regulação da Expressão Gênica/fisiologia , Histona Desacetilases/metabolismo , Neovascularização Fisiológica/fisiologia , Proteína Fosfatase 2/metabolismo , Grau de Desobstrução Vascular/fisiologia , Animais , Adesão Celular/fisiologia , Colágeno , Combinação de Medicamentos , Imunofluorescência , Regulação da Expressão Gênica/genética , Proteínas de Homeodomínio/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Processamento de Imagem Assistida por Computador , Laminina , Microscopia Confocal , Proteoglicanas , RNA Interferente Pequeno/genética , Grau de Desobstrução Vascular/genética , Peixe-Zebra
20.
Methods Mol Biol ; 1053: 283-305, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23860660

RESUMO

Protein phosphatases of the type 2A family (PP2A) represent a major fraction of cellular Ser/Thr phosphatase activity in any given human tissue. In this review, we describe how the holoenzymic nature of PP2A and the existence of several distinct PP2A composing subunits allow for the generation of multiple structurally and functionally different PP2A complexes, explaining why PP2A is involved in the regulation of so many diverse cell biological and physiological processes. Moreover, in human disease, most notably in several cancers and Alzheimer's Disease, PP2A expression and/or activity have been found significantly decreased, underscoring its important functions as a major tumor suppressor and tau phosphatase. Hence, several recent preclinical studies have demonstrated that pharmacological restoration of PP2A activity, as well as pharmacological PP2A inhibition, under certain conditions, may be of significant future therapeutic value.


Assuntos
Ativadores de Enzimas/farmacologia , Inibidores Enzimáticos/farmacologia , Proteína Fosfatase 2/química , Doença de Alzheimer/enzimologia , Doença de Alzheimer/metabolismo , Ativadores de Enzimas/química , Inibidores Enzimáticos/química , Holoenzimas/química , Holoenzimas/metabolismo , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/química , Isoenzimas/metabolismo , Neoplasias/enzimologia , Neoplasias/metabolismo , Proteína Fosfatase 2/antagonistas & inibidores , Proteína Fosfatase 2/metabolismo
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