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1.
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
2.
PLoS One ; 6(6): e21521, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21731772

RESUMO

BACKGROUND: Tau phosphorylation and dephosphorylation regulate in a poorly understood manner its physiological role of microtubule stabilization, and equally its integration in Alzheimer disease (AD) related fibrils. A specific phospho-pattern will result from the balance between kinases and phosphatases. The heterotrimeric Protein Phosphatase type 2A encompassing regulatory subunit PR55/Bα (PP2A(T55α)) is a major Tau phosphatase in vivo, which contributes to its final phosphorylation state. We use NMR spectroscopy to determine the dephosphorylation rates of phospho-Tau by this major brain phosphatase, and present site-specific and kinetic data for the individual sites including the pS202/pT205 AT8 and pT231 AT180 phospho-epitopes. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrate the importance of the PR55/Bα regulatory subunit of PP2A within this enzymatic process, and show that, unexpectedly, phosphorylation at the pT231 AT180 site negatively interferes with the dephosphorylation of the pS202/pT205 AT8 site. This inhibitory effect can be released by the phosphorylation dependent prolyl cis/trans isomerase Pin1. Because the stimulatory effect is lost with the dimeric PP2A core enzyme (PP2A(D)) or with a phospho-Tau T231A mutant, we propose that Pin1 regulates the interaction between the PR55/Bα subunit and the AT180 phospho-epitope on Tau. CONCLUSIONS/SIGNIFICANCE: Our results show that phosphorylation of T231 (AT180) can negatively influence the dephosphorylation of the pS202/pT205 AT8 epitope, even without an altered PP2A pool. Thus, a priming dephosphorylation of pT231 AT180 is required for efficient PP2A(T55α)-mediated dephosphorylation of pS202/pT205 AT8. The sophisticated interplay between priming mechanisms reported for certain Tau kinases and the one described here for Tau phosphatase PP2A(T55α) may contribute to the hyperphosphorylation of Tau observed in AD neurons.


Assuntos
Doença de Alzheimer/enzimologia , Peptidilprolil Isomerase/metabolismo , Proteína Fosfatase 2/metabolismo , Proteínas tau/metabolismo , Doença de Alzheimer/patologia , Sequência de Aminoácidos , Animais , Ciclina A/metabolismo , Quinase 2 Dependente de Ciclina/metabolismo , Epitopos/metabolismo , Humanos , Cinética , Dados de Sequência Molecular , Proteínas Mutantes/metabolismo , Peptidilprolil Isomerase de Interação com NIMA , Fosforilação , Multimerização Proteica , Coelhos , Proteínas tau/química
3.
Proc Natl Acad Sci U S A ; 108(17): 6957-62, 2011 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-21482799

RESUMO

Functional diversity of protein phosphatase 2A (PP2A) enzymes mainly results from their association with distinct regulatory subunits. To analyze the functions of one such holoenzyme in vivo, we generated mice lacking PR61/B'δ (B56δ), a subunit highly expressed in neural tissues. In PR61/B'δ-null mice the microtubule-associated protein tau becomes progressively phosphorylated at pathological epitopes in restricted brain areas, with marked immunoreactivity for the misfolded MC1-conformation but without neurofibrillary tangle formation. Behavioral tests indicated impaired sensorimotor but normal cognitive functions. These phenotypical characteristics were further underscored in PR61/B'δ-null mice mildly overexpressing human tau. PR61/B'δ-containing PP2A (PP2A(T61δ)) poorly dephosphorylates tau in vitro, arguing against a direct dephosphorylation defect. Rather, the activity of glycogen synthase kinase-3ß, a major tau kinase, was found increased, with decreased phosphorylation of Ser-9, a putative cyclin-dependent kinase 5 (CDK5) target. Accordingly, CDK5 activity is decreased, and its cellular activator p35, strikingly absent in the affected brain areas. As opposed to tau, p35 is an excellent PP2A(T61δ) substrate. Our data imply a nonredundant function for PR61/B'δ in phospho-tau homeostasis via an unexpected spatially restricted mechanism preventing p35 hyperphosphorylation and its subsequent degradation.


Assuntos
Encéfalo/enzimologia , Quinase 5 Dependente de Ciclina/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Dobramento de Proteína , Proteína Fosfatase 2/metabolismo , Tauopatias/enzimologia , Animais , Quinase 5 Dependente de Ciclina/genética , Quinase 3 da Glicogênio Sintase/genética , Glicogênio Sintase Quinase 3 beta , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Células PC12 , Fosforilação/genética , Proteína Fosfatase 2/genética , Ratos , Tauopatias/genética , Proteínas tau/genética , Proteínas tau/metabolismo
4.
Nat Cell Biol ; 12(9): 886-93, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20711181

RESUMO

When vertebrate cells exit mitosis various cellular structures are re-organized to build functional interphase cells. This depends on Cdk1 (cyclin dependent kinase 1) inactivation and subsequent dephosphorylation of its substrates. Members of the protein phosphatase 1 and 2A (PP1 and PP2A) families can dephosphorylate Cdk1 substrates in biochemical extracts during mitotic exit, but how this relates to postmitotic reassembly of interphase structures in intact cells is not known. Here, we use a live-cell imaging assay and RNAi knockdown to screen a genome-wide library of protein phosphatases for mitotic exit functions in human cells. We identify a trimeric PP2A-B55alpha complex as a key factor in mitotic spindle breakdown and postmitotic reassembly of the nuclear envelope, Golgi apparatus and decondensed chromatin. Using a chemically induced mitotic exit assay, we find that PP2A-B55alpha functions downstream of Cdk1 inactivation. PP2A-B55alpha isolated from mitotic cells had reduced phosphatase activity towards the Cdk1 substrate, histone H1, and was hyper-phosphorylated on all subunits. Mitotic PP2A complexes co-purified with the nuclear transport factor importin-beta1, and RNAi depletion of importin-beta1 delayed mitotic exit synergistically with PP2A-B55alpha. This demonstrates that PP2A-B55alpha and importin-beta1 cooperate in the regulation of postmitotic assembly mechanisms in human cells.


Assuntos
Mitose/fisiologia , Proteína Fosfatase 2/metabolismo , Interferência de RNA , beta Carioferinas/metabolismo , Divisão do Núcleo Celular/efeitos dos fármacos , Divisão do Núcleo Celular/fisiologia , Cromossomos/metabolismo , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/metabolismo , Flavonoides/farmacologia , Complexo de Golgi/metabolismo , Células HeLa , Histonas/metabolismo , Humanos , Processamento de Imagem Assistida por Computador/métodos , Interfase/fisiologia , Leupeptinas/farmacologia , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Mitose/efeitos dos fármacos , Modelos Biológicos , Fosforilação/fisiologia , Piperidinas/farmacologia , Ligação Proteica/fisiologia , Proteína Fosfatase 2/genética , RNA Interferente Pequeno/genética , Fuso Acromático/metabolismo , Transfecção , beta Carioferinas/genética
5.
Mol Cell ; 37(5): 633-42, 2010 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-20227368

RESUMO

The mammalian target of rapamycin (mTOR) pathway is activated by a variety of stimuli, including nutrients such as glucose and amino acids. The Ste20 family kinase MAP4K3 is regulated by amino acids and acts upstream of mTORC1. Here we investigate how MAP4K3 activity is regulated by amino acid sufficiency. We identify a transautophosphorylation site in the MAP4K3 kinase activation segment (Ser170) that is required for MAP4K3 activity and its activation of mTORC1 signaling. Following amino acid withdrawal, Ser170 is dephosphorylated via PP2A complexed to PR61 epsilon, a PP2A-targeting subunit. Inhibition of PR61 epsilon expression prevents MAP4K3 Ser170 dephosphorylation and impairs mTORC1 inhibition during amino acid withdrawal. We propose that during amino acid sufficiency Ser170-phosphorylated MAP4K3 activates mTORC1, but that upon amino acid restriction MAP4K3 preferentially interacts with PP2A(T61 epsilon), promoting dephosphorylation of Ser170, MAP4K3 inhibition, and, subsequently, inhibition of mTORC1 signaling.


Assuntos
Aminoácidos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteína Fosfatase 2/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal , Aminoácidos/deficiência , Linhagem Celular , Ativação Enzimática , Humanos , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Mutação , Fosforilação , Ligação Proteica , Proteína Fosfatase 2/genética , Proteínas Serina-Treonina Quinases/genética , Subunidades Proteicas , Proteínas/metabolismo , Interferência de RNA , Proteína Regulatória Associada a mTOR , Serina-Treonina Quinases TOR , Transfecção
6.
FASEB J ; 24(2): 538-47, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19825976

RESUMO

To elucidate novel cell biological functions of specific protein phosphatase 2A (PP2A) holoenzymes, we identified and biochemically characterized a complex between the Src homology 2 (SH2) domain-containing inositol polyphosphate 5-phosphatase 2 (SHIP2) and a PP2A holoenzyme comprising PR130/B''alpha1 as a regulatory subunit (PP2A(T130)) in several mammalian cell lines. PR130/B''alpha1 and SHIP2 partially colocalize in untreated HeLa cells, and both translocate to the cell membrane on epidermal growth factor (EGF) stimulation. Concomitantly, a transient EGF-dependent interaction of PR130/B''alpha1 with the EGF receptor (EGFR) was observed, whereas the SHIP2-PR130 interaction remained constitutive. As previously reported for SHIP2, RNA interference-mediated knockdown of PR130 in COS-7 cells resulted in increased EGF-induced proteasome-dependent EGFR degradation, and an increased interaction of EGFR with the E3 ligase c-Cbl. In concordance with faster EGFR clearance or desensitization, intrinsic EGFR kinase activity (phospho-Tyr-1068) and downstream protein kinase B and extracellular signal-regulated kinase/mitogen-activated protein kinase pathways were more rapidly inactivated in PR130-knockdown cells. Notably, these effects could be rescued by reintroduction of RNA interference-resistant Myc-PR130, excluding any off-target effect. These data highlight a novel biological role of the PP2A(T130) holoenzyme in EGF signaling through interaction with EGFR and the phosphatidylinositol (3,4,5)-trisphosphate 5-phosphatase SHIP2. This interaction may be of clinical relevance as dysfunction of EGF-mediated signaling has been linked to various human cancers.


Assuntos
Receptores ErbB/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Proteína Fosfatase 2/metabolismo , Subunidades Proteicas/metabolismo , Animais , Células COS , Chlorocebus aethiops , Fator de Crescimento Epidérmico/metabolismo , Fator de Crescimento Epidérmico/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/efeitos dos fármacos , Células HeLa , Humanos , Inositol Polifosfato 5-Fosfatases , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases , Proteínas Proto-Oncogênicas c-akt/fisiologia , Interferência de RNA , Transdução de Sinais/efeitos dos fármacos
7.
Biochem Biophys Res Commun ; 386(4): 676-81, 2009 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-19555667

RESUMO

Protein phosphatase 2A (PP2A) represents a family of multimeric serine/threonine phosphatases with pleiotropic roles in signal transduction. We previously described the functional analysis of two Ca(2+)-binding EF-hands in the PR72/B'' class of regulatory PP2A subunits. Now we report partial degradation of PR72/B"alpha2 and PR130/B"alpha1 into a 45-48kDa proteolysis-resistant fragment ('PR45') by the Ca(2+)-dependent protease m-calpain. This limited proteolysis is dependent on EF-hand integrity, independent of two PEST-domains, and highly specific as PP2A(C), PR65/A and representatives of PR55/B and PR61/B' subunit families are calpain-resistant. PR45 was also generated in staurosporine-induced apoptotic MCF7 cells in a calpain-dependent way. Calpain treatment weakens the PR72-core enzyme interaction, activates basal PP2A(T72) phosphatase activity and dramatically increases its sensitivity for and activation by polycations. This unique property can be exploited in a specific biochemical assay for these holoenzymes. We propose local calpain action in vivo may constitute a novel regulatory mechanism of these holoenzymes.


Assuntos
Calpaína/metabolismo , Proteína Fosfatase 2/metabolismo , Animais , Linhagem Celular , Humanos , Mutação , Proteína Fosfatase 2/genética , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Coelhos
8.
BMC Genomics ; 9: 393, 2008 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-18715506

RESUMO

BACKGROUND: Protein phosphatase 2A (PP2A) is a serine/threonine-specific phosphatase displaying vital functions in growth and development through its role in various signalling pathways. PP2A holoenzymes comprise a core dimer composed of a catalytic C and a structural A subunit, which can associate with a variable B-type subunit. The importance of the B-type subunits for PP2A regulation cannot be overestimated as they determine holoenzyme localisation, activity and substrate specificity. Three B-type subunit families have been identified: PR55/B, PR61/B' and PR72/B", of which the latter is currently the least characterised. RESULTS: We deduced the sequences and genomic organisation of the different murine PR72/B" isoforms: three genes encode nine isoforms, five of which are abundantly expressed and give rise to genuine PP2A subunits. Thereby, one novel subunit was identified. Using Northern blotting, we examined the tissue-specific and developmental expression of these subunits. All subunits are highly expressed in heart, suggesting an important cardiac function. Immunohistochemical analysis revealed a striated expression pattern of PR72 and PR130 in heart and skeletal muscle, but not in bladder smooth muscle. The subcellular localisation and cell cycle regulatory ability of several PR72/B" isoforms were determined, demonstrating differences as well as similarities. CONCLUSION: In contrast to PR55/B and PR61/B', the PR72/B" family seems evolutionary more divergent, as only two of the murine genes have a human orthologue. We have integrated these results in a more consistent nomenclature of both human and murine PR72/B" genes and their transcripts/proteins. Our results provide a platform for the future generation of PR72/B" knockout mice.


Assuntos
Proteínas de Ciclo Celular/genética , Biologia Computacional , Regulação da Expressão Gênica no Desenvolvimento , Proteína Fosfatase 2/genética , Processamento Alternativo , Sequência de Aminoácidos , Animais , Bases de Dados de Proteínas , Humanos , Isoenzimas/genética , Camundongos , Dados de Sequência Molecular , Músculo Esquelético/citologia , Miocárdio/citologia , Filogenia , Subunidades Proteicas/genética , Sondas RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
9.
Proc Natl Acad Sci U S A ; 105(12): 4727-32, 2008 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-18339811

RESUMO

Class IIa histone deacetylases (HDACs) act as key transcriptional regulators in several important developmental programs. Their activities are controlled via phosphorylation-dependent nucleocytoplasmic shuttling. Phosphorylation of conserved serine residues triggers association with 14-3-3 proteins and cytoplasmic relocalization of class IIa HDACs, which leads to the derepression of their target genes. Although a lot of effort has been made toward the identification of the inactivating kinases that phosphorylate class IIa HDAC 14-3-3 motifs, the existence of an antagonistic protein phosphatase remains elusive. Here we identify PP2A as a phosphatase responsible for dephosphorylating the 14-3-3 binding sites in class IIa HDACs. Interestingly, dephosphorylation of class IIa HDACs by PP2A is prevented by competitive association of 14-3-3 proteins. Using both okadaic acid treatment and RNA interference, we demonstrate that PP2A constitutively dephosphorylates the class IIa member HDAC7 to control its biological functions as a regulator of T cell apoptosis and endothelial cell functions. This study unravels a dynamic interplay among 14-3-3s, protein kinases, and PP2A and provides a model for the regulation of class IIa HDACs.


Assuntos
Apoptose , Histona Desacetilases/metabolismo , Neovascularização Fisiológica , Proteína Fosfatase 2/metabolismo , Linfócitos T/citologia , Linfócitos T/enzimologia , Proteínas 14-3-3/metabolismo , Apoptose/efeitos dos fármacos , Linhagem Celular , Citoplasma/efeitos dos fármacos , Citoplasma/enzimologia , Inibidores Enzimáticos/farmacologia , Humanos , Neovascularização Fisiológica/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Proteína Fosfatase 2/antagonistas & inibidores , Transporte Proteico/efeitos dos fármacos , RNA Interferente Pequeno/metabolismo , Proteínas Repressoras/metabolismo , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/enzimologia , Linfócitos T/efeitos dos fármacos
10.
Trends Biochem Sci ; 33(3): 113-21, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18291659

RESUMO

Protein phosphatase 2A (PP2A), a major phospho-serine/threonine phosphatase, is conserved throughout eukaryotes. It dephosphorylates a plethora of cellular proteins, including kinases and other signaling molecules involved in cell division, gene regulation, protein synthesis and cytoskeleton organization. PP2A enzymes typically exist as heterotrimers comprising catalytic C-, structural A- and regulatory B-type subunits. The B-type subunits function as targeting and substrate-specificity factors; hence, holoenzyme assembly with the appropriate B-type subunit is crucial for PP2A specificity and regulation. Recently, several biochemical and structural determinants have been described that affect PP2A holoenzyme assembly. Moreover, the effects of specific post-translational modifications of the C-terminal tail of the catalytic subunit indicate that a 'code' might regulate dynamic exchange of regulatory B-type subunits, thus affecting the specificity of PP2A.


Assuntos
Proteína Fosfatase 2/química , Proteína Fosfatase 2/metabolismo , Animais , Catálise , Holoenzimas/química , Holoenzimas/metabolismo , Humanos , Modelos Biológicos , Conformação Proteica , Estrutura Secundária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Especificidade por Substrato
11.
Exp Cell Res ; 314(1): 68-81, 2008 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-17803990

RESUMO

Reversible methylation of the protein phosphatase 2A catalytic subunit (PP2A(C)(1)) is an important regulatory mechanism playing a crucial role in the selective recruitment of regulatory B subunits. Here, we investigated the subcellular localization of leucine carboxyl methyltransferase (LCMT1) and protein phosphatase methylesterase (PME-1), the two enzymes catalyzing this process. The results show that PME-1 is predominantly localized in the nucleus and harbors a functional nuclear localization signal, whereas LCMT1 is underrepresented in the nucleus and mainly localizes to the cytoplasm, Golgi region and late endosomes. Indirect immunofluorescence with methylation-sensitive anti-PP2A(C) antibodies revealed a good correlation with the methylation status of PP2A(C), demethylated PP2A(C) being substantially nuclear. Throughout mitosis, demethylated PP2A(C) is associated with the mitotic spindle and during cytokinesis with the cleavage furrow. Overexpression of PME-1, but not of an inactive mutant, results in increased demethylation of PP2A(C) in the nucleus, whereas overexpression of a cytoplasmic PME-1 mutant lacking the NLS results in increased demethylation in the cytoplasm-in all cases, however, without any obvious functional consequences. PME-1 associates with an inactive PP2A population, regardless of its esterase activity or localization. We propose that stabilization of this inactive, nuclear PP2A pool is a major in vivo function of PME-1.


Assuntos
Hidrolases de Éster Carboxílico/metabolismo , Compartimento Celular/fisiologia , Núcleo Celular/enzimologia , Citoplasma/enzimologia , Proteína Fosfatase 2/metabolismo , Transporte Ativo do Núcleo Celular/fisiologia , Animais , Células COS , Núcleo Celular/ultraestrutura , Chlorocebus aethiops , Citoplasma/ultraestrutura , Endossomos/enzimologia , Endossomos/ultraestrutura , Complexo de Golgi/enzimologia , Complexo de Golgi/ultraestrutura , Células HeLa , Humanos , Metilação , Mitose/fisiologia , Sinais de Localização Nuclear , Proteína O-Metiltransferase/metabolismo , Proteína Fosfatase 2/química , Transporte Proteico/fisiologia , Transdução de Sinais/fisiologia , Fuso Acromático/metabolismo
12.
Proc Natl Acad Sci U S A ; 104(50): 19867-72, 2007 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-18056802

RESUMO

Inactivation of maturation-promoting factor [(MPF) Cdk1/Cyclin B] is a key event in the exit from mitosis. Although degradation of Cyclin B is important for MPF inactivation, recent studies indicate that Cdk1 phosphorylation and inactivation occur before Cyclin B degradation and, therefore, also may be important steps in the exit from mitosis. Cdk1 activity is controlled by the Cdc25C phosphatase, which is turned on at the G(2)/M transition to catalyze Cdk1 activation. PP2A:B56delta is a negative regulator of Cdc25C during interphase. We show here that PP2A:B56delta also regulates Cdc25C at mitosis. Failure of PP2A:B56delta to dephosphorylate Cdc25C at mitosis results in prolonged hyperphosphorylation and activation of Cdc25C, causing persistent dephosphorylation and, hence, activation of Cdk1. This constitutive activation of Cdc25C and Cdk1 leads to a delayed exit from mitosis. Consistent with Cdk1 as a major biological target of B56delta, stable knockdown and germ-line mouse KO of B56delta leads to compensatory transcriptional up-regulation of Wee1 kinase to oppose the Cdc25C activity and permit cell survival. These observations place PP2A:B56delta as a key upstream regulator of Cdk1 activity upon exit from mitosis.


Assuntos
Proteína Quinase CDC2/metabolismo , Mitose , Proteína Fosfatase 2/metabolismo , Fosfatases cdc25/metabolismo , Animais , Proteína Quinase CDC2/genética , Linhagem Celular , Ativação Enzimática , Regulação Enzimológica da Expressão Gênica , Humanos , Mesotelina , Camundongos , Ligação Proteica , Proteína Fosfatase 2/genética , Regulação para Cima
13.
J Biol Chem ; 282(37): 26971-26980, 2007 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-17635907

RESUMO

Protein phosphatase 2A (PP2A) is a family of multifunctional serine/threonine phosphatases all composed of a catalytic C, a structural A, and a regulatory B subunit. Assembly of the complex with the appropriate B subunit forms the key to the functional specificity and regulation of PP2A. Emerging evidence suggests a crucial role for methylation and phosphorylation of the PP2A C subunit in this process. In this study, we show that PP2A C subunit methylation was not absolutely required for binding the PR61/B' and PR72/B'' subunit families, whereas binding of the PR55/B subunit family was determined by methylation and the nature of the C-terminal amino acid side chain. Moreover mutation of the phosphorylatable Tyr(307) or Thr(304) residues differentially affected binding of distinct B subunit family members. Down-regulation of the PP2A methyltransferase LCMT1 by RNA interference gradually reduced the cellular amount of methylated C subunit and induced a dynamic redistribution of the remaining methylated PP2A(C) between different PP2A trimers consistent with their methylation requirements. Persistent knockdown of LCMT1 eventually resulted in specific degradation of the PR55/B subunit and apoptotic cell death. Together these results establish a crucial foundation for understanding PP2A regulatory subunit selection.


Assuntos
Fosfoproteínas Fosfatases/química , Animais , Apoptose , Células COS , Domínio Catalítico , Sobrevivência Celular , Chlorocebus aethiops , Células HeLa , Humanos , Metilação , Metiltransferases/fisiologia , Fosfoproteínas Fosfatases/fisiologia , Fosforilação , Proteína Fosfatase 2 , Relação Estrutura-Atividade
14.
Proc Natl Acad Sci U S A ; 104(23): 9876-81, 2007 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-17535922

RESUMO

In dopaminoceptive neurons, dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32) plays a central role in integrating the effects of dopamine and other neurotransmitters. Phosphorylation of DARPP-32 at Thr-34 by protein kinase A results in inhibition of protein phosphatase 1 (PP1), and phosphorylation at Thr-75 by Cdk5 (cyclin-dependent kinase 5) results in inhibition of protein kinase A. Dephosphorylation at Thr-34 involves primarily the Ca(2+)-dependent protein phosphatase, PP2B (calcineurin), whereas dephosphorylation of Thr-75 involves primarily PP2A, the latter being subject to control by both cAMP- and Ca(2+)-dependent regulatory mechanisms. In the present study, we have investigated the mechanism of Ca(2+)-dependent regulation of Thr-75 by PP2A. We show that the PR72 (or B'' or PPP2R3A) regulatory subunit of PP2A is highly expressed in striatum. Through the use of overexpression and down-regulation by using RNAi, we show that PP2A, in a heterotrimeric complex with the PR72 subunit, mediates Ca(2+)-dependent dephosphorylation at Thr-75 of DARPP-32. The PR72 subunit contains two Ca(2+) binding sites formed by E and F helices (EF-hands 1 and 2), and we show that the former is necessary for the ability of PP2A activity to be regulated by Ca(2+), both in vitro and in vivo. Our studies also indicate that the PR72-containing form of PP2A is necessary for the ability of glutamate acting at alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid and NMDA receptors to regulate Thr-75 dephosphorylation. These studies further our understanding of the complex signal transduction pathways that regulate DARPP-32. In addition, our studies reveal an alternative intracellular mechanism whereby Ca(2+) can activate serine/threonine phosphatase activity.


Assuntos
Fosfoproteína 32 Regulada por cAMP e Dopamina/metabolismo , Neurônios/metabolismo , Proteína Fosfatase 2/metabolismo , Cálcio/metabolismo , Linhagem Celular , Humanos , Immunoblotting , Imunoprecipitação , Oligonucleotídeos , Fosforilação , Subunidades Proteicas/metabolismo , Interferência de RNA
15.
Mol Cell ; 23(3): 413-24, 2006 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-16885030

RESUMO

PTPA, an essential and specific activator of protein phosphatase 2A (PP2A), functions as a peptidyl prolyl isomerase (PPIase). We present here the crystal structures of human PTPA and of the two yeast orthologs (Ypa1 and Ypa2), revealing an all alpha-helical protein fold that is radically different from other PPIases. The protein is organized into two domains separated by a groove lined by highly conserved residues. To understand the molecular mechanism of PTPA activity, Ypa1 was cocrystallized with a proline-containing PPIase peptide substrate. In the complex, the peptide binds at the interface of a peptide-induced dimer interface. Conserved residues of the interdomain groove contribute to the peptide binding site and dimer interface. Structure-guided mutational studies showed that in vivo PTPA activity is influenced by mutations on the surface of the peptide binding pocket, the same mutations that also influenced the in vitro activation of PP2Ai and PPIase activity.


Assuntos
Peptidilprolil Isomerase/química , Fosfoproteínas Fosfatases/química , Proteínas/química , Proteínas de Saccharomyces cerevisiae/química , Sequência de Aminoácidos , Sítios de Ligação/genética , Cristalografia por Raios X , Dimerização , Ativação Enzimática , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Modelos Moleculares , Dados de Sequência Molecular , Mutação/genética , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Peptídeos/química , Prolina/química , Conformação Proteica , Proteína Fosfatase 2 , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Proteínas/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de Aminoácidos
16.
FEBS Lett ; 580(15): 3631-7, 2006 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-16764867

RESUMO

A trimeric protein phosphatase 2A (PP2A(T55)) composed of the catalytic (PP2Ac), structural (PR65/A), and regulatory (PR55/B) subunits was isolated from rabbit skeletal muscle by thiophosphorylase affinity chromatography, and contained two additional proteins of 54 and 55 kDa, respectively. The 54 kDa protein was identified as eukaryotic translation termination factor 1 (eRF1) and as a PP2A interacting protein. The 55 kDa protein is now identified as nucleoredoxin (NRX). The formation of a complex between GST-NRX, PP2A(C) and PP2A(D) was demonstrated by pull-down experiments with purified forms of PP2A, and by immunoprecipitation of HA-tagged NRX expressed in HEK293 cells complexed endogenous PP2A subunits. Analysis of PP2A activity in the presence of GST-NRX showed that NRX competed with polycations for both stimulatory and inhibitory effects on different forms of PP2A.


Assuntos
Proteínas Nucleares/metabolismo , Oxirredutases/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Dimerização , Regulação da Expressão Gênica , Humanos , Camundongos , Dados de Sequência Molecular , Peso Molecular , Proteínas Nucleares/química , Proteínas Nucleares/genética , Oxirredutases/química , Oxirredutases/genética , Fosfoproteínas Fosfatases/isolamento & purificação , Ligação Proteica , Proteína Fosfatase 2 , Coelhos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
17.
J Biol Chem ; 281(10): 6349-57, 2006 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-16380387

RESUMO

The protein phosphatase 2A (PP2A) phosphatase activator (PTPA) is an essential protein involved in the regulation of PP2A and the PP2A-like enzymes. In this study we demonstrate that PTPA and its yeast homologues Ypa1 and Ypa2 can induce a conformational change in some model substrates. Using these model substrates in different assays with and without helper proteases, this isomerase activity is similar to the isomerase activity of FKBP12, the human cyclophilin A, and one of its yeast homologs Cpr7 but dissimilar to the isomerase activity of Pin1. However, neither FKBP12 nor Cpr7 can reactivate the inactive form of PP2A. Therefore, PTPA belongs to a novel peptidyl-prolyl cis/trans-isomerase (PPIase) family. The PPIase activity of PTPA correlates with its activating activity since both are stimulated by the presence of Mg2+ATP, and a PTPA mutant (Delta208-213) with 400-fold less activity in the activation reaction of PP2A also showed almost no PPIase activity. The point mutant Asp205 --> Gly (in Ypa1) identified this amino acid as essential for both activities. Moreover, PTPA dissociates the inactive form from the complex with the PP2A methylesterase. Finally, Pro190 in the catalytic subunit of PP2A (PP2AC) could be identified as the target Pro isomerized by PTPA/Mg2+ATP since among the 14 Pro residues present in 12 synthesized peptides representing the microenvironments of these prolines in PP2AC, only Pro190 could be isomerized by PTPA/Mg2+ATP. This Pro190 is present in a predicted loop structure near the catalytic center of PP2AC and, if mutated into a Phe, the phosphatase is inactive and can no longer be activated by PTPA/Mg2+ATP.


Assuntos
Peptidilprolil Isomerase/fisiologia , Fosfoproteínas Fosfatases/metabolismo , Proteínas/fisiologia , Trifosfato de Adenosina/fisiologia , Animais , Células COS , Domínio Catalítico , Chlorocebus aethiops , Ciclofilina A/genética , Ciclofilina A/fisiologia , Peptidil-Prolil Isomerase F , Ciclofilinas/genética , Ciclofilinas/fisiologia , Humanos , Cinética , Magnésio/fisiologia , Família Multigênica , Mutagênese Sítio-Dirigida , Peptidilprolil Isomerase de Interação com NIMA , Peptidilprolil Isomerase/genética , Peptidilprolil Isomerase/metabolismo , Fosfoproteínas Fosfatases/antagonistas & inibidores , Prolina/genética , Prolina/metabolismo , Proteína Fosfatase 2 , Proteínas/genética , Coelhos , Especificidade por Substrato , Proteína 1A de Ligação a Tacrolimo/genética , Proteína 1A de Ligação a Tacrolimo/fisiologia
18.
Enzymes ; 24: 303-24, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-26718045

RESUMO

PP2A has been shown to be methylated at the C-terminal leucine residue of the catalytic subunit by a specific 38 kDa methyltransferase (LCMT1) and demethylated by a specific 44-kDa methylesterase (PME-1). This reversible methylation does not seem to drastically change the PP2A activity but is shown to be a modulating factor in the binding of the third regulatory subunit. The structure of LCMT1 is solved and a model for the catalysis of the methylation reaction is presented. By purifying the PP2A-methylesterase, inactive dimeric (PP2AiD) and trimeric (PP2AiT55) holoenzymes were found to be associated with PME-1. Activation of this inactive complex is possible by the action of a ubiquitous and highly conserved activatory protein, PTPA. The function of PME-1in this system seems to be independent of its demethylating activity. A large proportion of cellular PP2A is found methylated and the subject of regulation. Aberrant (de)methylation seems to be involved in the causes of diseases such as Alzheimer's disease and diabetes.

19.
FEBS Lett ; 579(16): 3392-6, 2005 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-15936019

RESUMO

A direct interaction of the regulatory domain (R domain) of the cystic fibrosis transmembrane conductance regulator protein (CFTR) with PR65, a regulatory subunit of the protein phosphatase 2A (PP2A), was shown in yeast two hybrid, pull-down and co-immunoprecipitation experiments. The R domain could be dephosphorylated by PP2A in vitro. Overexpression of the interacting domain of PR65 in Caco-2 cells, as well as treatment with okadaic acid, showed a prolonged deactivation of the chloride channel. Taken together our results show a direct and functional interaction between CFTR and PP2A.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Sequência de Aminoácidos , Animais , Células CACO-2 , Humanos , Imunoprecipitação , Dados de Sequência Molecular , Proteína Fosfatase 2 , Estrutura Terciária de Proteína , Subunidades Proteicas/metabolismo , Técnicas do Sistema de Duplo-Híbrido
20.
Curr Opin Genet Dev ; 15(1): 34-41, 2005 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-15661531

RESUMO

PP2A is one of the few serine/threonine-specific phosphatases in the cell, and its complex structure and regulation guarantees its many different functions. Some viruses have chosen to target this enzyme system in order to manage the host cell machinery for their own profit and to program cells into a malignant state. Suppression of PR61/B'gamma, a specific third regulatory subunit of PP2A, can substitute for the viral SV40 protein small t antigen in causing tumorigenic transformation of several human cell lines -- provided that telomerase, SV40 large T antigen and oncogenic Ras are also present. Accumulation of c-Myc seems to be the common denominator.


Assuntos
Fosfoproteínas Fosfatases/fisiologia , Proteínas Supressoras de Tumor/fisiologia , Animais , Ciclo Celular/genética , Ciclo Celular/fisiologia , Humanos , Neoplasias/enzimologia , Neoplasias/genética , Neoplasias/metabolismo , Fosfoproteínas Fosfatases/genética , Proteínas Supressoras de Tumor/genética
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