蛋白磷酸酶4的组成与主要功能

在蛋白质的可逆磷酸化过程中,蛋白激酶和蛋白磷酸酶有着同等重要的作用。近年来,人们逐渐把研究的重点转移到以往关注甚少的蛋白磷酸酶家族上。蛋白磷酸酶4(PP4或PPX)是蛋白磷酸酶2A(PP2A)家族的重要成员之一,它与多个调节亚基形成多种复合体参与诸多重要的细胞进程,如中心体的成熟、剪接体复合体的组装、多个细胞信号通路的调节以及DNA损伤修复的调节等多个事件。现对PP4的组成、活性调节及已知的生物学功能作简要介绍。     

植物PP2C蛋白磷酸酶ABA信号转导及逆境胁迫调控机制研究进展

蛋白磷酸酶(protein phosphatase,PP)是蛋白质可逆磷酸化调节机制中的关键酶,而PP2C磷酸酶是一类丝氨酸/苏氨酸残基蛋白磷酸酶,是高等植物中最大的蛋白磷酸酶家族,包含76个家族成员,广泛存在于生物体中。迄今为止,在植物体内已经发现了4种PP2C蛋白磷酸酶。蛋白激酶和蛋白磷酸酶协同催化蛋白质可逆磷酸化,在植物体内信号转导和生理代谢中起着重要的调节作用,蛋白质的磷酸化几乎存在于所有的信号转导途径中。大量研究表明,PP2Cs参与多条信号转导途径,包括PP2C参与ABA调控,对干旱、低温、高盐等逆境胁迫的响应,参与植物创伤和种子休眠或萌发等信号途径,其调控机制不同,但酶催化活性都依赖于Mg2+或Mn2+的浓度。植物PP2C蛋白的C端催化结构域高度保守,而N端功能各异。文中还综述了高等植物PP2C的分类、结构、ABA受体与PP2Cs蛋白互作、PP2C基因参与ABA信号途径以及其他逆境信号转导途径的研究进展。     

蛋白磷酸酶2A在真菌细胞中的研究进展

蛋白质可逆的磷酸化修饰在真菌细胞生命活动中发挥着重要作用。磷酸化与去磷酸化过程相互协调。蛋白质中磷酸丝氨酸/苏氨酸位点的去磷酸化反应主要由蛋白磷酸酶2A (Protein Phosphatase2A,PP2A)负责催化。PP2A由催化亚基、调节亚基与结构亚基组合成有活性的三聚体全酶形式发挥功能。本文以模式真菌酿酒酵母、裂殖酵母与人类条件致病菌白色念珠菌为例,总结了PP2A家族成员在真菌细胞中的研究进展及去磷酸化作用调控真菌细胞生命活动的重要性,分析了PP2A调控真菌生命活动尚需解析的作用机制,并提出了可能的研究思路。     

植物蛋白磷酸酶2C(PP2C)及其在信号转导中的作用

蛋白磷酸酶(protein phosphatase,PP)是蛋白质可逆磷酸化调节机制中的关键酶,蛋白磷酸酶2C(PP2C)是蛋白磷酸酶的一个分支。文章介绍了PP2C的结构及其在信号转导中的研究进展。     

PP2A在运动抗阿尔茨海默病进程中的机制研究

Tau蛋白过度磷酸化是AD发病的重要原因,促进脑中p-Tau蛋白的脱磷酸化进程是运动抗AD的重要途径。PP2A是重要的蛋白磷酸酶,对p-Tau蛋白的脱磷酸化有重要作用。有关PP2A介导运动抗AD的Tau蛋白磷酸化机制研究尚不多见。现从Tau蛋白与AD研究、PP2A与AD研究、运动与AD研究、PP2A与运动抗AD研究等方面,系统阐述PP2A在介导运动抗AD进程中的蛋白磷酸化机制,为探明运动抗AD的Tau蛋白途径及运动促进健康的蛋白质修饰机制研究提供参考。     

蛋白磷酸酶PP2A主要通过B’家族调节亚基介导Dishevelled2蛋白的去磷酸化

Dishevelled2(Dvl2)是Wnt信号通路中的关键蛋白因子且受到剧烈的磷酸化调控。蛋白磷酸酶2A(PP2A)是Dvl2的一种磷酸酶,参与Dvl2的去磷酸化调控。PP2A有多达16种调节亚基,决定着PP2A的底物特异性,但参与调节Dvl2去磷酸化的PP2A调节亚基尚未有全面研究。该文在一种细胞系中,通过siRNA逐一敲低PP2A调节亚基基因表达,分析了所有调节亚基在Dvl2磷酸化调控中的参与程度。结果显示,多种PP2A调节亚基参与Dvl2去磷酸化,其中B’家族全部成员均有参与,起到主要调控作用。细胞共定位和蛋白互作实验结果同样印证PP2A调节亚基B’家族成员参与Dvl2蛋白的磷酸化调控。该研究明确了对Dvl2蛋白去磷酸化起调控作用的PP2A调节亚基,有助于了解PP2A调节亚基的细胞生物学功能以及与底物的关系。     

蛋白酪氨酸磷酸酶SHP-2与疾病的相关性研究进展

蛋白酪氨酸磷酸酶家族由130多种蛋白酪氨酸磷酸酶组成,它们和蛋白质酪氨酸激酶家族一起调控蛋白质中酪氨酸残基的磷酸化以及去磷酸化的动态平衡,它们的活性直接决定细胞内蛋白质的磷酸化水平的高低。SHP-2是蛋白酪氨酸磷酸酶家族的一员,在各种细胞和组织中均有广泛的表达,参与多个信号传导通路,介导细胞的生长、分化、迁移、粘附及凋亡等。SHP-2的表达异常会导致多种疾病的产生,但是相关综述较少,同时未见文献报道其在胶质瘤中的作用,因此本文简要介绍SHP-2的结构、功能、信号传导,并阐述了SHP-2与常见疾病的关系。     

微囊藻毒素LR对人HL7702细胞蛋白磷酸酶2A的影响

微囊藻毒素能显著抑制蛋白磷酸酶2A的活性,是公认的一类肝毒素。为进一步研究微囊藻毒素作用于离体细胞后细胞中PP2A相关指标的变化,实验用不同浓度的MC-LR对人正常肝细胞HL7702染毒24h后检测细胞中PP2A亚基蛋白和mRNA水平,并检测3、6、12和24h作用后细胞内PP2A的酶活性。结果发现,除B55α蛋白表达有明显的下降趋势且1000 nmol/L组有显著性降低外,其他亚基的蛋白没有明显改变;15种PP2A亚基mRNA水平在100 nmol/L组都显著升高,而500和1000 nmol/L组分别有7种和6种亚基的mRNA表现出显著性降低。用不同浓度MC-LR染毒6h后,细胞内PP2A酶活有显著性降低,并且呈现剂量依赖性关系,而3、12和24h染毒后酶活性没有明显变化。以上结果表明:一定浓度的MC-LR作用24h后影响了HL7702细胞PP2A相关亚基的转录,但没有明显改变蛋白质水平;MC-LR对细胞内PP2A的酶活抑制作用与染毒时间密切相关。       

蛋白磷酸酶PP2A的结构及其肿瘤抑制因子功能

蛋白磷酸酶在细胞的生命活动中起着十分重要的作用,蛋白磷酸酶2A(protein phosphatase 2A, PP2A)作为蛋白磷酸酶家族中十分重要的一员,它几乎与所有真核细胞的生命活动都有密不可分的关系．2006年,PP2A核心酶和全酶晶体结构的陆续破解对于深入了解PP2A自身的结构和亚基之间的相互作用,以及其与结合蛋白作用的机制都有重大的影响．随着PP2A与肿瘤相关性的一系列新研究成果的不断涌现,PP2A在肿瘤发生和细胞迁移中也彰显出十分关键的作用．重点介绍PP2A的组成与结构、催化亚基的特殊修饰、亚基之间的相互作用关系以及PP2A作为一种新的肿瘤抑制因子的生物学功能．     

高等植物中蛋白磷酸酶2C的结构与功能

蛋白质磷酸化／去磷酸化是生物信号级联传递的重要方式之一,主要通过生化性质互为对立的蛋白激酶和蛋白磷酸酶实现。蛋白磷酸酶2C(PP2C)是蛋白磷酸酶的一个分支,其生化性质、蛋白质组成与结构都和其他磷酸酶显著不同,但都在生物信号传递中扮演重要角色。高等植物中PP2C广泛参与脱落酸(ABA)的各种信号途径,包括ABA诱导的种子萌发／休眠、保卫细胞及离子通道调控和气孔关闭、逆境胁迫等。PP2C也多样地参与植物创伤反应、生长发育以及抗病性等各个途径。作为大多数信号途径的负调控因子,PP2C能直接与激酶结合,与其他调控蛋白结合,以及直接与DNA结合调控相关基因的表达。     

Regulation of protein phosphatase 2A by hydrogen peroxide and glutathionylation

The regulation of protein phosphatase 2A (PP2A) and protein threonine phosphorylation by H(2)O(2) was determined in Caco-2 cell monolayer. Incubation with H(2)O(2) (20 microM) resulted in threonine phosphorylation of a cluster of proteins at the molecular mass range of 170-250 kDa. PKC activity and plasma membrane localization of several isoforms of PKC were not affected by H(2)O(2). However, H(2)O(2) reduced 80-85% of okadaic acid-sensitive protein phosphatase activity. Immunocomplex protein phosphatase assay demonstrated that H(2)O(2) reduced the activity of PP2A, but not that of PP2C or PP1. Oxidized glutathione inhibited PP2A activity in plasma membranes prepared from Caco-2 cells and the phosphatase activity of an isolated PP2A. PP2A activity was also inhibited by N-ethylmaleimide, iodoacetamide, and p-chloromercuribenzoate. Inhibition of PP2A by oxidized glutathione was reversed by reduced glutathione. Glutathione also restored the PP2A activity in plasma membranes isolated from H(2)O(2)-treated Caco-2 cell monolayer. These results indicate that PP2A activity can be regulated by glutathionylation, and that H(2)O(2) inhibits PP2A in Caco-2 cells, which may involve glutathionylation of PP2A.     

Autophosphorylation of ataxia-telangiectasia mutated is regulated by protein phosphatase 2A

Ionizing radiation induces autophosphorylation of the ataxia-telangiectasia mutated (ATM) protein kinase on serine 1981; however, the precise mechanisms that regulate ATM activation are not fully understood. Here, we show that the protein phosphatase inhibitor okadaic acid (OA) induces autophosphorylation of ATM on serine 1981 in unirradiated cells at concentrations that inhibit protein phosphatase 2A-like activity in vitro. OA did not induce gamma-H2AX foci, suggesting that it induces ATM autophosphorylation by inactivation of a protein phosphatase rather than by inducing DNA double-strand breaks. In support of this, we show that ATM interacts with the scaffolding (A) subunit of protein phosphatase 2A (PP2A), that the scaffolding and catalytic (C) subunits of PP2A interact with ATM in undamaged cells and that immunoprecipitates of ATM from undamaged cells contain PP2A-like protein phosphatase activity. Moreover, we show that IR induces phosphorylation-dependent dissociation of PP2A from ATM and loss of the associated protein phosphatase activity. We propose that PP2A plays an important role in the regulation of ATM autophosphorylation and activity in vivo.     

Pertussis Toxin Modification of PC12 Cells Inhibits a Protein Phosphatase 2A-Like Phosphatase

Abstract: We have found that modification of rat PC12 cells with pertussis toxin resulted in an ∼50% inhibition of a protein phosphatase 2A-like phosphatase. Protein phosphatase 2A (PP2A) is a major cellular serine/threonine-specific protein phosphatase. Treatment of extracts from pertussis toxin-modified PC12 cells with either immobilized alkaline phosphatase or Ca²⁺ reversed this inhibition. Reactivation of the PP2A-like phosphatase in Ca²⁺ appears to result from the dephosphorylation of a protein by the Ca²⁺/calmodulin-dependent protein phosphatase calcineurin. The PP2A-like phosphatase in extracts from pertussis toxin-modified PC12 cells eluted from a Mono Q column at a higher ionic strength than did the PP2A-like phosphatase in extracts from control cells. After incubation in Ca²⁺, the PP2A-like phosphatase in extracts from pertussis toxin-modified cells eluted from a Mono Q column at the same ionic strength as did the PP2A-like phosphatase in extracts from control cells. These results indicate that the effect of pertussis toxin on this PP2A-like activity results from the phosphorylation of either one of the subunits of the PP2A-like phosphatase or a protein that when phosphorylated binds to and inhibits this phosphatase. Pertussis toxin modification did not result in the phosphorylation of the catalytic subunit of PP2A. Because phosphorylation regulates the activities of many enzymes and cell surface receptors, a pertussis toxin-induced decrease in PP2A activity could alter signaling pathways and other cellular processes in which G proteins are not directly involved.     

Role for protein phosphatase 2A in the regulation of Calu-3 epithelial Na+-K+-2Cl-, type 1 co-transport function

Activity of Na+-K+-2Cl- co-transport (NKCC1) in epithelia is thought to be highly regulated through phosphorylation and dephosphorylation of the transporter. Previous functional studies from this laboratory suggested a role for protein phosphatase 2A (PP2A) as a serine/threonine protein phosphatase involved in the regulation of mammalian tracheal epithelial NKCC1. We expand on these studies to characterize serine/threonine protein phosphatase(s) necessary for regulation of NKCC1 function and the interaction of the phosphatase(s) with proteins associated with NKCC1. NKCC1 activity was measured as bumetanide-sensitive 86Rb uptake or basolateral to apical 86Rb flux in primary cultures of human tracheal epithelial cells or in Calu-3 airway epithelial cells grown on Transwell filter inserts. Preincubation with 0.1 nm okadaic acid, a PP2A > phosphatase 1 (PP1) inhibitor, increased NKCC1 activity 3.5-fold in human tracheal epithelial cells and 4.1-fold in Calu-3 cells. Calyculin, a PP1 > PP2A inhibitor, did not alter NKCC1 activity or percent bumetanide-sensitive flux. The effect of OA was dose-dependent with an IC50 of 0.4 nm. The alpha1-adrenergic agonist methoxamine increased NKCC1 activity and transiently increased PP2A activity 3.8-fold but did not alter PP1 activity. OA augmented methoxamine-dependent stimulation of NKCC1 activity. PP1, PP2A, and PP2C but not PP2B were detected in lysates from Calu-3 cells by immunoblot analysis. PP1 was not detected in immunoprecipitates of NKCC1 and vice versa. PP2A co-immunoprecipitated with NKCC1 and protein kinase C-delta (PKC-delta) and was pulled down by a recombinant N terminus of NKCC1 consisting of amino acids 1-286. One novel finding is co-precipitation of STE20-related proline-alanine-rich kinase, a regulatory kinase for NKCC1, with PP2A and PKC-delta. The results suggest a model of actin serving as a scaffold for binding and association of PKC-delta, PP2A, and STE20-related proline-alanine-rich kinase. The role of the complex of serine/threonine protein kinases and a protein phosphatase is probably the maintenance of optimal phosphorylation of NKCC1 coincident with its physiological function in epithelial absorption and secretion.     

Protein Phosphatase Methyl-Esterase PME-1 Protects Protein Phosphatase 2A from Ubiquitin/Proteasome Degradation

Protein phosphatase 2A (PP2A) is a conserved essential enzyme that is implicated as a tumor suppressor based on its central role in phosphorylation-dependent signaling pathways. Protein phosphatase methyl esterase (PME-1) catalyzes specifically the demethylation of the C-terminal Leu309 residue of PP2A catalytic subunit (PP2Ac). It has been shown that PME-1 affects the activity of PP2A by demethylating PP2Ac, but also by directly binding to the phosphatase active site, suggesting loss of PME-1 in cells would enhance PP2A activity. However, here we show that PME-1 knockout mouse embryonic fibroblasts (MEFs) exhibit lower PP2A activity than wild type MEFs. Loss of PME-1 enhanced poly-ubiquitination of PP2Ac and shortened the half-life of PP2Ac protein resulting in reduced PP2Ac levels. Chemical inhibition of PME-1 and rescue experiments with wild type and mutated PME-1 revealed methyl-esterase activity was necessary to maintain PP2Ac protein levels. Our data demonstrate that PME-1 methyl-esterase activity protects PP2Ac from ubiquitin/proteasome degradation.     

Characterization of the interaction of polyomavirus middle T antigen with type 2A protein phosphatase.

Two cellular proteins of 36 and 63 kDa which bind the small T and middle T antigens of polyomavirus recently have been identified as the catalytic and regulatory subunits of the phosphoserine/threonine-specific type 2A protein phosphatase (PP2A). We report here the presence of phosphoseryl phosphatase activity associated with polyomavirus small T and middle T antigens in immunoprecipitates prepared from virus-infected and transformed cells. Phosphatase activity was also found associated with middle T-antigen mutants, some of which had been defined previously to associate with 36- and 63-kDa cellular proteins. Middle T-antigen-associated phosphatase activity was sensitive to okadaic acid and microcystin-LR, inhibitors of PP2A, and insensitive to inhibitor 1 or 2, orthovanadate, or EDTA. Using antiserum specific for the catalytic subunit of PP2A, we found that unlike the majority of PP2A, middle T-antigen-bound PP2A was membrane associated. However, no gross change in the amount, activity, or localization of PP2A could be attributed to middle T-antigen expression in transformed cells. Anti-PP2A antibodies coprecipitated a 63-kDa protein from normal cells and in addition coprecipitated middle T antigen, 60- and 61-kDa proteins (identified as src family members), and an 81-kDa protein from middle T-antigen-transformed cells. Furthermore, we detected protein kinase activity in PP2A immunoprecipitates and protein phosphatase activity in src immune complexes from extracts of middle T-antigen-transformed, but not normal, cells. These results reinforce the notion that at least a portion of middle T antigen bridges a protein kinase with a protein phosphatase.     

Inhibition of Src by direct interaction with protein phosphatase 2A

In this study, we report that Src kinase is inhibited by protein phosphatase 2A (PP2A), a serine/threonine phosphatase. We carried out experiments in vitro using purified PP2A (AC dimer) and full-length v-Src or truncated forms of v-Src. The inhibition of v-Src by PP2A is concentration- and time-dependent. Addition of okadaic acid, a PP2A phosphatase inhibitor, abolished the PP2A-dependent inhibition of v-Src. When experiments were carried out at 4 degrees C under conditions where PP2A activity is inhibited, Src activity was unaffected by the presence of PP2A, suggesting that PP2A binding alone is insufficient to block Src activity. These results imply that PP2A activity is essential for inhibition of v-Src. We also demonstrate that PP2A binds to the catalytic and the regulatory domains of v-Src.     

The primary identification of a calcineurin A subunit-like protein in plants

Many types of serine/threonine protein phosphatase have been cloned and characterized in plants, such as Type-1 serine/threonine protein phosphatase (PP1), Type-2A serine/threonine protein phosphatase (PP2A), Type-2C serine/threonine protein phosphatase (PP2C). However no Type-2B serine/threonine protein phosphatase (PP2B, calcineurin), or calcineurin A subunit-like protein (CaNAL), has been identified. We detected protein phosphatase activity in mixtures of CaM-binding proteins from three plants (Nicotiana tabacum, Brassica oleracea and Arabidopsis thaliana). Two-dimensional electrophoresis (2-D) and Western blot analysis with an anti-rat CNA antibody revealed a small protein of 60 kDa that we believe is a CaNAL. The isoelectric point (pI) of this protein in N. tabacum was approximately 5.69. The protein phosphatase activity in the mixture of CaM-binding proteins from N. tabacum was regulated by Ca²⁺ and Calmodulin (CaM) with either RII peptides or pNPP as substrate. The immunosuppressive drugs, CsA and FK506, also inhibited the protein phosphatase activity significantly.     

Protein phosphatase 2A1 is the major enzyme in vertebrate cell extracts that dephosphorylates several physiological substrates for cyclin-dependent protein kinases.

Okadaic acid (2 nM) inhibited by 80-90% the protein phosphatase activities in diluted extracts of rat liver, human fibroblasts, and Xenopus eggs acting on three substrates (high mobility group protein-I(Y), caldesmon and histone H1) phosphorylated by a cyclin-dependent protein kinase (CDK) suggesting that a type-2A phosphatase was responsible for dephosphorylating each protein. This result was confirmed by anion exchange chromatography of rat liver and Xenopus extracts, which demonstrated that the phosphatases acting on these substrates coeluted with the two major species of protein phosphatase 2A, termed PP2A1 and PP2A2. When matched for activity toward glycogen phosphorylase, PP2A1 was five- to sevenfold more active than PP2A2 and 35-fold to 70-fold more active than the free catalytic subunit (PP2Ac) toward the three CDK-labeled substrates. Protein phosphatases 1, 2B, and 2C accounted for a negligible proportion of the activity toward each substrate under the assay conditions examined. The results suggest that PP2A1 is the phosphatase that dephosphorylates a number of CDK substrates in vivo and indicate that the A and B subunits that are associated with PP2Ac in PP2A1 accelerate the dephosphorylation of CDK substrates, while suppressing the dephosphorylation of most other proteins. The possibility that PP2A1 activity is regulated during the cell cycle is discussed.     

Brain PP2A is modified by thiol-disulfide exchange and intermolecular disulfide formation

The regulation of protein phosphatase 2A (PP2A) activity by thiol-disulfide exchange and resulting formation of an intermolecular disulfide was examined following exposure of a rat brain soluble fraction to a biotinylated derivative of the model disulfide HPDP (HPDP-biotin) which would be expected to label reactive protein thiols with a disulfide-linked biotin. The results show that a low concentration (500 microM) of HPDP-biotin produced substantial inhibition of PP2A activity and promoted the binding of the catalytic subunit of PP2A to an immobilized avidin-affinity column. Both the inhibition of PP2A activity and the binding of PP2A to the avidin column were reversed by treatment with the disulfide reducing agent dithiothreitol (DTT). Furthermore, the specific activity of PP2A was up to 7-fold higher in the DTT-eluted fractions from the avidin-affinity column than in the soluble fraction. These findings demonstrate directly that PP2A is susceptible to reversible inhibitory modification by thiol-disulfide exchange and provide mechanistic support for the emerging view that PP2A is an oxidant-sensitive protein phosphatase.