植物中14-3-3蛋白的主要功能

14-3-3蛋白家族广泛存在于真核生物中,序列高度保守。主要以同源或异源二聚体形式存在,可以同时与两个靶蛋白或者与一个靶蛋白的两个结构域相互作用,通过与靶蛋白上的一小段共有序列的磷酸化丝氨酸/苏氨酸残基结合来发挥其调控功能。本文综述了植物中的14-3-3蛋白及其主要功能,并重点综述了14-3-3蛋白对植物基本碳、氮代谢的调控。     

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。     

Akt基因的研究进展

Akt是一种丝氨酸/苏氨酸蛋白激酶,对细胞代谢、细胞增殖、细胞凋亡、细胞周期等多种生物学过程进行调控。本文主要综述了Akt的结构、调控机制、靶蛋白及其功能。     

细胞周期依赖性激酶4(Cdk4)在肿瘤发生发展中的作用

细胞周期依赖性激酶4(cdk4)是丝氨酸/苏氨酸激酶家族的成员,调控细胞周期G1期的进程.Cdk4与周期蛋白D(cyclin D)结合形成复合物,在G1期的演进中起重要作用,一旦出现失调就可能导致癌症的发生,并且也有一系列的内在和外在的信号调控着这个复合物.Cdk4以及它的调控因子在肿瘤的发生和转移中都显示了重要的作用.     

逆境下拟南芥ABA信号途径负调控因子的研究进展

ABA信号途径的主要负调控因子蛋白磷酸酶2C(protein phosphatase 2C,PP2C)是一类丝氨酸/苏氨酸蛋白磷酸酶(protein Ser/Thr phosphatases,PSP),为ABA信号传导途径下游的关键组分.拟南芥中PP2C主要包括ABI1、ABI2、HAB1、AHG3和PP2CA,它们通过改变ABA信号的强弱等调控植物的胁迫应答.该文主要对国内外有关PP2C家族的组成以及在逆境胁迫下负调控ABA信号途径中的调控机制和应答特征等方面的研究进展进行综述.     

蛋白磷酸酶PPM1A的研究进展

PPM1A,又称PP2Cα,即镁离子依赖的蛋白磷酸酶1A,是丝氨酸/苏氨酸蛋白磷酸酶家族一员,该家族被认为是真核细胞压力应答通路中必需的负向调控因子。近年来的研究结果表明,PPM1A可与多种蛋白结合使其去磷酸化,广泛调控如细胞生长、细胞应激、免疫反应和肿瘤形成等众多生命活动。本文对PPM1A的结构、活性调控和作用底物做一个简要的综述,以期对PPM1A有一个全面的了解进而有助于后期的进一步研究。     

14-3-3蛋白家族的调控机制和生物学功能

14-3-3蛋白家族在真核细胞中广泛表达并高度保守,它们主要以同源／异源二聚体形式存在,可以同时与两个靶蛋白或一个靶蛋白的两个结构域相互作用。14-3-3蛋白通过磷酸化丝氨酸／苏氨酸介导和靶蛋白结合,从而发挥其调控功能。现对14-3-3蛋白的识别序列、与配体相互作用的特点,及其在细胞周期、凋亡、信号转导、线粒体／叶绿体前体蛋白跨膜转运中的调控机制和发挥的生物学功能进行综述。     

OST1调控植物气孔运动和逆境响应的研究进展

SnRK2(SNF1-related protein kinase 2)家族在调控植物应答和抵御逆境方面发挥着重要作用。Open Stomata 1(OST1)/SnRK2.6是该家族的成员,具有典型的丝氨酸/苏氨酸蛋白激酶保守域,并主要在保卫细胞中表达。在逆境胁迫下,蛋白磷酸酶2C解除对OST1蛋白激酶的抑制,随后OST1蛋白激酶启动对下游信号组分的调控作用并引起气孔运动。本研究综述了OST1蛋白激酶的结构特征,主要概述OST1蛋白激酶与蛋白磷酸酶、转录因子和离子通道的调控关系,最后对相关的研究进行了展望。     

β肾上腺素受体的结构与功能域

β肾上腺素受体具有视紫红质样结构,包括由膜两侧亲水环相互联结7个疏水性跨膜α螺旋结构,N端无信号序列而含有2个N-糖基化位点,C端富含丝氨酸和苏氨酸残基.7个跨膜结构构成配基结合位点.β受体细胞膜内侧环状序列形成两亲α螺旋结构,与G蛋白相互作用.C端及第3个内侧环的丝氨酸及苏氨酸残基构成受体磷酸化位点,参与受体功能调控.     

急性缺氧和急性低糖对脑片tau蛋白磷酸化的影响

为探讨急性缺氧对tau蛋白磷酸化的影响,将Wistar大鼠脑片进行不同时间的缺氧培养后,对tau蛋白的磷酸化状态及相关磷酸酯酶的活性和表达进行检测.结果显示,急性缺氧使tau蛋白多个丝氨酸位点磷酸化水平下降,蛋白磷酸酯酶～2A(PP-2A)的活性升高,其催化亚单位表达上调,而蛋白磷酸酯酶-1(PP-1)的活性及催化亚单位表达均无明显改变.该研究结果表明:急性缺氧可能通过蛋白磷酸酯酶-2A的上调而使tau蛋白多个丝氨酸位点发生去磷酸化作用.     

Crystal structure of the protein serine/threonine phosphatase 2C at 2.0 A resolution.

Protein phosphatase 2C (PP2C) is a Mn2+- or Mg2+-dependent protein Ser/Thr phosphatase that is essential for regulating cellular stress responses in eukaryotes. The crystal structure of human PP2C reveals a novel protein fold with a catalytic domain composed of a central beta-sandwich that binds two manganese ions, which is surrounded by alpha-helices. Mn2+-bound water molecules at the binuclear metal centre coordinate the phosphate group of the substrate and provide a nucleophile and general acid in the dephosphorylation reaction. Our model presents a framework for understanding not only the classical Mn2+/Mg2+-dependent protein phosphatases but also the sequence-related domains of mitochondrial pyruvate dehydrogenase phosphatase, the Bacillus subtilus phosphatase SpoIIE and a 300-residue domain within yeast adenyl cyclase. The protein architecture and deduced catalytic mechanism are strikingly similar to the PP1, PP2A, PP2B family of protein Ser/Thr phosphatases, with which PP2C shares no sequence similarity, suggestive of convergent evolution of protein Ser/Thr phosphatases.     

Dephosphorylation of the focal adhesion protein VASP in vitro and in intact human platelets

The focal adhesion protein VASP, a possible link between signal transduction pathways and the microfilament system, is phosphorylated by both cAMP- and cGMP-dependent protein kinases in vitro and in intact cells. Here, the analysis of VASP dephosphorylation by the serine/threonine protein phosphatases (PP) PP1, PP2A, PP2B and PP2C in vitro is reported. The phosphatases differed in their selectivity with respect to the dephosphorylation of individual VASP phosphorylation sites. Incubation of human platelets with okadaic acid, a potent inhibitor of PP1 and PP2A, caused the accumulation of phosphorylated VASP indicating that the phosphorylation status of VASP in intact cells is regulated to a major extent by serine/ threonine protein phosphatases. Furthermore, the accumulation of phosphorylated cAMP-dependent protein kinase substrate(s) appears to account for inhibitory effects of okadaic acid on platelet function.     

Type 2C protein phosphatases in fungi

Type 2C Ser/Thr phosphatases are a remarkable class of protein phosphatases, which are conserved in eukaryotes and involved in a large variety of functional processes. Unlike in other Ser/Thr phosphatases, the catalytic polypeptide is not usually associated with regulatory subunits, and functional specificity is achieved by encoding multiple isoforms. For fungi, most information comes from the study of type 2C protein phosphatase (PP2C) enzymes in Saccharomyces cerevisiae, where seven PP2C-encoding genes (PTC1 to -7) with diverse functions can be found. More recently, data on several Candida albicans PP2C proteins became available, suggesting that some of them can be involved in virulence. In this work we review the available literature on fungal PP2Cs and explore sequence databases to provide a comprehensive overview of these enzymes in fungi.     

The complement of protein phosphatase catalytic subunits encoded in the genome of Arabidopsis

Reversible protein phosphorylation is critically important in the modulation of a wide variety of cellular functions. Several families of protein phosphatases remove phosphate groups placed on key cellular proteins by protein kinases. The complete genomic sequence of the model plant Arabidopsis permits a comprehensive survey of the phosphatases encoded by this organism. Several errors in the sequencing project gene models were found via analysis of predicted phosphatase coding sequences. Structural sequence probes from aligned and unaligned sequence models, and all-against-all BLAST searches, were used to identify 112 phosphatase catalytic subunit sequences, distributed among the serine (Ser)/threonine (Thr) phosphatases (STs) of the protein phosphatase P (PPP) family, STs of the protein phosphatase M (PPM) family (protein phosphatases 2C [PP2Cs] subfamily), protein tyrosine (Tyr) phosphatases (PTPs), low-M(r) protein Tyr phosphatases, and dual-specificity (Tyr and Ser/Thr) phosphatases (DSPs). The Arabidopsis genome contains an abundance of PP2Cs (69) and a dearth of PTPs (one). Eight sequences were identified as new protein phosphatase candidates: five dual-specificity phosphatases and three PP2Cs. We used phylogenetic analyses to infer clustering patterns reflecting sequence similarity and evolutionary ancestry. These clusters, particularly for the largely unexplored PP2C set, will be a rich source of material for plant biologists, allowing the systematic sampling of protein function by genetic and biochemical means.     

Protein phosphatase 2C (PP2C) function in higher plants

In the past few years, molecular cloning studies have revealed the primary structure of plant protein serine/threonine phosphatases. Two structurally distinct families, the PP1/PP2A family and the PP2C family, are present in plants as well as in animals. This review will focus on the plant PP2C family of protein phosphatases. Biochemical and molecular genetic studies in Arabidopsis have identified PP2C enzymes as key players in plant signal transduction processes. For instance, the ABI1/ABI2 PP2Cs are central components in abscisic acid (ABA) signal transduction. Arabidopsis mutants containing a single amino acid exchange in ABI1 or ABI2 show a reduced response to ABA. Another member of the PP2C family, kinase-associated protein phosphatase (KAPP), appears to be an important element in some receptor-like kinase (RLK) signalling pathways. Finally, an alfalfa PP2C acts as a negative regulator of a plant mitogen-activated protein kinase (MAPK) pathway. Thus, the plant PP2Cs function as regulators of various signal transduction pathways.     

The specificity of extracellular signal-regulated kinase 2 dephosphorylation by protein phosphatases

The extracellular signal-regulated protein kinase 2 (ERK2) is the founding member of a family of mitogen-activated protein kinases (MAPKs) that are central components of signal transduction pathways for cell proliferation, stress responses, and differentiation. The MAPKs are unique among the Ser/Thr protein kinases in that they require both Thr and Tyr phosphorylation for full activation. The dual phosphorylation of Thr-183 and Tyr-185 in ERK2 is catalyzed by MAPK/ERK kinase 1 (MEK1). However, the identity and relative activity of protein phosphatases that inactivate ERK2 are less well established. In this study, we performed a kinetic analysis of ERK2 dephosphorylation by protein phosphatases using a continuous spectrophotometric enzyme-coupled assay that measures the inorganic phosphate produced in the reaction. Eleven different protein phosphatases, many previously suggested to be involved in ERK2 regulation, were compared, including tyrosine-specific phosphatases (PTP1B, CD45, and HePTP), dual specificity MAPK phosphatases (VHR, MKP3, and MKP5), and Ser/Thr protein phosphatases (PP1, PP2A, PP2B, PP2C alpha, and lambda PP). The results provide biochemical evidence that protein phosphatases display exquisite specificity in their substrate recognition and implicate HePTP, MKP3, and PP2A as ERK2 phosphatases. The fact that ERK2 inactivation could be carried out by multiple specific phosphatases shows that signals can be integrated into the pathway at the phosphatase level to determine the cellular response to external stimuli. Important insights into the roles of various protein phosphatases in ERK2 kinase signaling are obtained, and further analysis of the mechanism by which different protein phosphatases recognize and inactivate MAPKs will increase our understanding of how this kinase family is regulated.     

Role of protein phosphatases in the activation of CFTR (ABCC7) by genistein and bromotetramisole

Genistein and bromotetramisole(Br-t) strongly activate cystic fibrosis transmembrane conductanceregulator (CFTR; ABCC7) chloride channels on Chinese hamster ovarycells and human airway epithelial cells. We have examined the possiblerole of phosphatases in stimulation by these drugs using patch-clampand biochemical methods. Genistein inhibited the spontaneous rundown ofchannel activity that occurs after membrane patches are excised fromcAMP-stimulated cells but had no effect on purified protein phosphatasetype 1 (PP1), PP2A, PP2B, PP2C, or endogenous phosphatases when assayed as [³²P]PO₄ release from prelabeled casein,recombinant GST-R domain fusion protein, or immunoprecipitatedfull-length CFTR. Br-t also slowed rundown of CFTR channels, but, inmarked contrast to genistein, it did inhibit all four proteinphosphatases tested. Half-maximal inhibition of PP2A and PP2C wasobserved with 0.5 and 1.5 mM Br-t, respectively. Protein phosphataseswere also sensitive to (+)-p-Br-t, a stereoisomer of Br-tthat does not inhibit alkaline phosphatases. Br-t appeared to actexclusively through phosphatases since it did not affect CFTR channelsin patches that had low apparent endogenous phosphatase activity (i.e.,those lacking spontaneous rundown). We conclude that genistein and Br-tact through different mechanisms. Genistein stimulates CFTR withoutinhibiting phosphatases, whereas Br-t acts by inhibiting amembrane-associated protein phosphatase (probably PP2C) that presumablyallows basal phosphorylation to accumulate.

     

The role of PP5 and PP2C in cardiac health and disease

Protein phosphatases are important, for example, as functional antagonists of β-adrenergic stimulation of the mammalian heart. While β-adrenergic stimulations increase the phosphorylation state of regulatory proteins and therefore force of contraction in the heart, these phosphorylations are reversed and thus force is reduced by the activity of protein phosphatases. In this context the role of PP5 and PP2C is starting to unravel. They do not belong to the same family of phosphatases with regard to sequence homology, many similarities with regard to location, activation by lipids and putative substrates have been worked out over the years. We also suggest which pathways for regulation of PP5 and/or PP2C described in other tissues and not yet in the heart might be useful to look for in cardiac tissue. Both phosphatases might play a role in signal transduction of sarcolemmal receptors in the heart. Expression of PP5 and PP2C can be increased by extracellular stimuli in the heart. Because PP5 is overexpressed in failing animal and human hearts, and because overexpression of PP5 or PP2C leads to cardiac hypertrophy and KO of PP5 leads to cardiac hypotrophy, one might argue for a role of PP5 and PP2C in heart failure. Because PP5 and PP2C can reduce, at least in vitro, the phosphorylation state of proteins thought to be relevant for cardiac arrhythmias, a role of these phosphatases for cardiac arrhythmias is also probable. Thus, PP5 and PP2C might be druggable targets to treat important cardiac diseases like heart failure, cardiac hypertrophy and cardiac arrhythmias.     

Role of Type 2C Protein Phosphatases in Growth Regulation and in Cellular Stress Signaling

ABSTRACT

A number of interesting features, phenotypes, and potential clinical applications have recently been ascribed to the type 2C family of protein phosphatases. Thus far, 16 different PP2C genes have been identified in the human genome, encoding (by means of alternative splicing) for at least 22 different isozymes. Virtually ever since their discovery, type 2C phosphatases have been predominantly linked to cell growth and to cellular stress signaling. Here, we provide an overview of the involvement of type 2C phosphatases in these two processes, and we show that four of them (PP2Cα, PP2Cβ, ILKAP, and PHLPP) can be expected to function as tumor suppressor proteins, and one as an oncoprotein (PP2Cδ /Wip1). In addition, we demonstrate that in virtually all cases in which they have been linked to the stress response, PP2Cs act as inhibitors of cellular stress signaling. Based on the vast amount of experimental evidence obtained thus far, it therefore seems justified to conclude that type 2C protein phosphatases are important physiological regulators of cell growth and of cellular stress signaling.