Reconstruction of spatial structure of plant protein phosphatase type-1 and -2A in complex with okadaic acid

The homology modeling, based on known temple structures of Homo sapiens protein phosphatase type-1 and -2A was implemented. The spatial structures of the human protein phosphatases and their plant homologs from Arabidopsis thaliana was predicted. The quality of models was confirmed by conformational analysis and root mean square deviations. The sites of okadaic acid binding in molecules of plant protein phosphatases (type-1 and -2A) were proved by the data of comparative analysis and molecular dynamics.     

Cdk1 and okadaic acid-sensitive phosphatases control assembly of nuclear pore complexes in Drosophila embryos

Disassembly and reassembly of the nuclear pore complexes (NPCs) is one of the major events during open mitosis in higher eukaryotes. However, how this process is controlled by the mitotic machinery is not clear. To investigate this we developed a novel in vivo model system based on syncytial Drosophila embryos. We microinjected different mitotic effectors into the embryonic cytoplasm and monitored the dynamics of disassembly/reassembly of NPCs in live embryos using fluorescently labeled wheat germ agglutinin (WGA) or in fixed embryos using electron microscopy and immunostaining techniques. We found that in live embryos Cdk1 activity was necessary and sufficient to induce disassembly of NPCs as well as their cytoplasmic mimics: annulate lamellae pore complexes (ALPCs). Cdk1 activity was also required for keeping NPCs and ALPCs disassembled during mitosis. In agreement recombinant Cdk1/cyclin B was able to induce phosphorylation and dissociation of nucleoporins from the NPCs in vitro. Conversely, reassembly of NPCs and ALPCs was dependent on the activity of protein phosphatases, sensitive to okadaic acid (OA). Our findings suggest a model where mitotic disassembly/reassembly of the NPCs is regulated by a dynamic equilibrium of Cdk1 and OA-sensitive phosphatase activities and provide evidence that mitotic phosphorylation mediates disassembly of the NPC.     

Fluorescence studies on the interaction of inhibitor 2 and okadaic acid with the catalytic subunit of type 1 phosphoprotein phosphatases.

Phosphatase inhibitor 2 was mutagenized and expressed in Escherichia coli to produce a protein with a single cysteinyl residue at position 129. The newly introduced sulfhydryl group was labeled with a maleimide derivative of coumarin (CPM). The resulting fluorescent inhibitor 2 molecule (CPM-I2) retains biological activity and binds to the catalytic subunit of type 1 phosphatase (PP1-C) with a Kd similar to the Ki of native I2 (2-3 nM). Fluorescence anisotropy data indicate that kinase FA (glycogen synthase kinase 3) does not dissociate the CPM-I2.PP1-C complex but rather causes a conformational change in the I2 molecule that is retained even after the CPM-I2 is displaced by an excess of native I2. The fluorescence data presented here also indicate that okadaic acid and I2 are competitive for binding to PP1-C, even after kinase FA treatment of the CPM-I2.PP1-C complex.     

Differentiation induction in human breast tumor cells by okadaic acid and related inhibitors of protein phosphatases 1 and 2A.

Okadaic acid (OA), an inhibitor of protein phosphatases 1 and 2A, induces differentiation in human MCF-7, AU-565, and MB-231 breast tumor cells. In MCF-7 cells, OA elicited within 5 min an increase in the levels of a set of phosphorylated cellular proteins, within hours expression of the early response genes junB, c-jun, and c-fos, and within days manifestation of differentiation. Differentiation was also induced by two related protein phosphatase inhibitors, but not by an inactive OA derivative or by an inhibitor that penetrates epithelial cells poorly. These results indicate that OA and related agents can induce tumor breast cell differentiation, and this induction is correlated with their ability to inhibit PPH 1 and 2A.     

Importance of the C28-C38 hydrophobic domain of okadaic acid for potent inhibition of protein serine-threonine phosphatases 1 and 2A

Okadaic acid is a potent inhibitor of select serine/threonine protein phosphatases. The importance of the C28-C38 hydrophobic domain of okadaic acid for inhibition of PP1 and PP2A was investigated. The hydrophobic domain is required but not sufficient for potent inhibition, and it also contributes to differential inhibition between PP1 and PP2A.     

PP1/PP2A phosphatases inhibitors okadaic acid and calyculin A block ERK5 activation by growth factors and oxidative stress

Okadaic acid is an inhibitor of the protein Ser/Thr phosphatases PP1 and PP2A, which blocks the activation of extracellular signal-regulated protein kinase 5 (ERK5), a member of the MAP kinase family activated by growth factors and several types of stressors. The blocking of ERK5 activation by okadaic acid was observed in HeLa cells exposed to epidermal growth factor and H(2)O(2) as well as in PC12 cells stimulated by nerve growth factor and H(2)O(2). Calyculin A, another PP1 and PP2A inhibitor, behaved similarly although these compounds are not structurally related. This suggests that either PP1 or PP2A or both are necessary for ERK5 activation. Protein kinase C (PKC) acts as a negative regulator of the ERK5 activation pathway, however our data suggest that the effects of PKC and the phosphatase are unrelated.     

Crystal structure of the tumor-promoter okadaic acid bound to protein phosphatase-1

Protein phosphatase-1 (PP1) plays a key role in dephosphorylation in numerous biological processes such as glycogen metabolism, cell cycle regulation, smooth muscle contraction, and protein synthesis. Microorganisms produce a variety of inhibitors of PP1, which include the microcystin class of inhibitors and okadaic acid, the latter being the major cause of diarrhetic shellfish poisoning and a powerful tumor promoter. We have determined the crystal structure of the molecular complex of okadaic acid bound to PP1 to a resolution of 1.9 A. This structure reveals that the acid binds in a hydrophobic groove adjacent to the active site of the protein and interacts with basic residues within the active site. Okadaic acid exhibits a cyclic structure, which is maintained via an intramolecular hydrogen bond. This is reminiscent of other macrocyclic protein phosphatase inhibitors. The inhibitor-bound enzyme shows very little conformational change when compared with two other PP1 structures, except in the inhibitor-sensitive beta12-beta13 loop region. The selectivity of okadaic acid for protein phosphatases-1 and -2A but not PP-2B (calcineurin) may be reassessed in light of this study.     

Temporal and spatial regulation of the phosphatidate phosphatases lipin 1 and 2

Lipins are the founding members of a novel family of Mg(2+)-dependent phosphatidate phosphatases (PAP1 enzymes) that play key roles in fat metabolism and lipid biosynthesis. Despite their importance, there is still little information on how their activity is regulated. Here we demonstrate that the functions of lipin 1 and 2 are evolutionarily conserved from unicellular eukaryotes to mammals. The two lipins display distinct intracellular localization in HeLa M cells, with a pool of lipin 2 exhibiting a tight membrane association. Small interfering RNA-mediated silencing of lipin 1 leads to a dramatic decrease of the cellular PAP1 activity in HeLa M cells, whereas silencing of lipin 2 leads to an increase of lipin 1 levels and PAP1 activity. Consistent with their distinct functions in HeLa M cells, lipin 1 and 2 exhibit reciprocal patterns of protein expression in differentiating 3T3-L1 adipocytes. Lipin 2 levels increase in lipin 1-depleted 3T3-L1 cells without rescuing the adipogenic defects, whereas depletion of lipin 2 does not inhibit adipogenesis. Finally, we show that the PAP1 activity of both lipins is inhibited by phosphorylation during mitosis, leading to a decrease in the cellular PAP1 activity during cell division. We propose that distinct and non-redundant functions of lipin 1 and 2 regulate lipid production during the cell cycle and adipocyte differentiation.     

Induction of nuclear factor-kappa B and the human immunodeficiency virus long terminal repeat by okadaic acid, a specific inhibitor of phosphatases 1 and 2A

We have used a specific phosphatase inhibitor, okadaic acid, to examine the role of two phosphatases, PP1 and PP2A, in the induction of NF-kappa B and the long terminal repeat of the human immunodeficiency virus type 1 (HIV-LTR). Treatment of Jurkat cells with okadaic acid induced NF-kappa B in nuclear extracts. The rate of induction by okadaic acid was delayed compared to the induction of NF-kappa B by phorbol myristate acetate (PMA). The induction of NF-kappa B by okadaic acid was enhanced by cycloheximide or phytohemagglutinin (PHA). In contrast to PMA, okadaic acid appeared to induce NF-kappa B independently of protein kinase C (PKC). That the NF-kappa B induced by okadaic acid was functional was demonstrated by the marked increase in CAT activity that occurred in Jurkat, BJA-B, and U251 cells that were transfected with HIV-LTR-CAT and treated with okadaic acid. The increase in CAT activity triggered by okadaic acid was dependent on the presence of the NF-kappa B sites in the long terminal repeat of HIV as assessed by deletion and mutation analysis. Similarly to its effect on the induction of NF-kappa B, PHA added together with okadaic acid resulted in a further increase in CAT activity. Somewhat surprisingly, the addition of PMA inhibited the increase in CAT activity in response to okadaic acid, which suggests that the activation of PKC may also induce inhibitory factors.(ABSTRACT TRUNCATED AT 250 WORDS)     

S-phase arrest by reactive nitrogen species is bypassed by okadaic acid, an inhibitor of protein phosphatases PP1/PP2A

In mammalian cells DNA damage activates a checkpoint that halts progression through S phase. To determine the ability of nitrating agents to induce S-phase arrest, mouse C10 cells synchronized in S phase were treated with nitrogen dioxide (NO(2)) or SIN-1, a generator of reactive nitrogen species (RNS). SIN-1 or NO(2) induced S-phase arrest in a dose- and time-dependent manner. As for the positive controls adozelesin and cisplatin, arrest was accompanied by phosphorylation of ATM kinase; dephosphorylation of pRB; decreases in RF-C, cyclin D1, Cdc25A, and Cdc6; and increases in p21. Comet assays indicated that RNS induce minimal DNA damage. Moreover, in a cell-free replication system, nuclei from cells treated with RNS were able to support control levels of DNA synthesis when incubated in cytosolic extracts from untreated cells, whereas nuclei from cells treated with cisplatin were not. Induction of phosphatase activity may represent one mechanism of RNS-induced arrest, for the PP1/PP2A phosphatase inhibitor okadaic acid inhibited dephosphorylation of pRB; prevented decreases in the levels of RF-C, cyclin D1, Cdc6, and Cdc25A; and bypassed arrest by SIN-1 or NO(2), but not cisplatin or adozelesin. Our studies suggest that RNS may induce S-phase arrest through mechanisms that differ from those elicited by classical DNA-damaging agents.     

Interactions of okadaic acid with insulin action in hepatocytes: role of protein phosphatases in insulin action.

The regulation of carbohydrate metabolism involves changes in the phosphorylation state of enzymes. We used okadaic acid, a potent inhibitor of protein phosphatases type 2A (IC50 0.05-2 nM) and type 1 (IC50 10-20 nM) to determine the role of these phosphatases in the control of carbohydrate metabolism by insulin in rat hepatocytes. In the absence of insulin, okadaic acid caused total inhibition of glycogen synthesis at 100 nM and half-maximal inhibition at 8-9 nM. In the presence of insulin, lower concentrations of okadaic acid (to which type 2A phosphatases are sensitive) were effective at inhibiting glycogen synthesis. 2.5 nM okadaic acid caused total inhibition of the 2-fold stimulation of glycogen synthesis by insulin but had no effect on the basal unstimulated rate of glycogen synthesis. This suggests the involvement of type 2A protein phosphatases in the stimulation of glycogen synthesis by insulin. Okadaic acid (5 nM), partially suppressed but did not abolish the increase in glucokinase mRNA levels caused by insulin, indicating that dephosphorylation mechanisms may be involved in the control of glucokinase mRNA levels by insulin. It is concluded that activation of protein phosphatases type 1 and/or type 2A by insulin may have a widespread role in the control of glucose metabolism at various sites.     

A new model of the tautomycin-PP1 complex that is not analogous to the corresponding okadaic acid structure

A revised model of PP1-tautomycin (TM) complex suggests that this toxin does not bind in a conformation analogous to its structural cousin okadaic acid (OA), as has been assumed, but instead more resembles the mode of binding adopted by calyculin. This model rationalizes the unexpected potency of a truncated TM analogue lacking the bicyclic ketal common to TM and OA.     

The role of acid phosphatases in plant phosphorus metabolism

Hydrolysis of phosphate esters is a critical process in the energy metabolism and metabolic regulation of plant cells. This review summarizes the characteristics and putative roles of plant acid phosphatase (APase). Although immunologically closely related, plant APases display remarkable heterogeneity with regards to their kinetic and molecular properties, and subcellular location. The secreted APases of roots and cell cultures are relatively non-specific enzymes that appear to be important in the hydrolysis and mobilization of P_i from extracellular phosphomonoesters for plant nutrition. Intracellular APases are undoubtedly involved in the routine utilization of P_i reserves or other P_i-containing compounds. A special class of intracellular APase exists that demonstrate a clear-cut (but generally nonabsolute) substrate selectivity. These APases are hypothesized to have distinct metabolic functions and include: phytase, phosphoglycolate phosphatase, 3-phosphoglycerate phosphatase, phosphoenolpyruvate phosphatase, and phosphotyrosyl-protein phosphatase. APase expression is regulated by a variety of developmental and environmental factors. P_i starvation induces de novo synthesis of extra- and intracellular APases in cell cultures as well as in whole plants. Recommendations are made to achieve uniformity in the analyses of the different APase isoforms normally encountered within and between different plant tissues.     

The complete primary structure of calcineurin A, a calmodulin binding protein homologous with protein phosphatases 1 and 2A

A complementary DNA (cDNA) clone encoding the catalytic subunit of calcineurin (calcineurin A) has been isolated from a rat brain cDNA library. The primary structure of the cDNA consists of 2,337 nucleotides including the entire coding region for 521 amino acids, and the calculated molecular mass is 58,643 Da. The calcineurin A is strikingly homologous to protein phosphatases 1 and 2A, approximately 50% of the amino acids over an internal 250-residue region between residues 78 and 329 being identical. Twenty four amino acid-residue region between residues 391 and 414 shows the consensus structural features for a calmodulin-binding domain. These data suggest that the allosteric character of this chimeric enzyme is generated by gene fusion of two separate protein families.     

Cantharidin, another natural toxin that inhibits the activity of serine/threonine protein phosphatases types 1 and 2A

Cantharidin, a natural toxicant of blister beetles, is a strong inhibitor of protein phosphatases types 1(PP1) and 2A (PP2A). Like okadaic acid, cantharidin inhibits the activity of the purified catalytic subunit of PP2A (IC₅₀ = 0.16 μM) at a lower concentration than that of PPI (IC₅₀ = 1.7 μM) and only inhibits the activity of protein phosphatase type 2B (PP2B) at high concentrations. Dose-inhibition studies conducted with whole cell homogenates indicate that cantharidin also inhibits the native forms of these enzymes. Thus, cantharidin, which is economical and readily available, may be useful as an additional probe for studying the functions of serine/threonine protein phosphatases.     

山西植物功能型划分及其空间格局

随着全球气候变化的加剧,作为沟通陆地生态系统与气候变化的桥梁,植物功能型(Plant Functional Types,PFTs)越来越受到生态学家的关注。PFTs不仅是简化生态系统复杂性的有效工具,而且可将植物的生理生态过程、生物物理特征及物候变化等引入到动态植被模型中,研究气候变化下的植被反应及其反馈机制。为了在区域尺度上研究气候变化和植被反应,基于"生态-外貌"原则,依据植物特征(如生长型、叶的性状)及其对水分、温度的需求,结合区域的气候与地理条件,对山西植被进行植物功能型的划分,并在此基础上对其空间格局进行分析。结果表明:(1)山西植被可划分为19类植物功能型(其中包括4类栽培作物功能型),分别是:寒温性常绿针叶林、温性常绿针叶林、寒温性落叶针叶林、温性落叶阔叶林、高寒落叶灌丛、温性落叶灌丛、多年生禾草草原、多年生禾草草丛、多年生禾草草甸、多年生莎草草甸、多年生杂类草草原、多年生杂类草草丛、多年生杂类草草甸。1年生杂类草草甸、多年生豆科草原、果树、一年一熟栽培作物、一年二熟栽培作物和二年三熟栽培作物。植物功能型的划分和分布与山西植被区划有较好的一致性,基本反映了植物固有特征及其对水热条件的需求。(2)农作物在山西占有较大比重,占植被类型面积的53.15%,森林类型以温性常绿针叶林和温性落叶阔叶林为主,灌丛类型以温性落叶灌丛为主,草本类型中多年生禾草草丛占较大比例,占草本类型面积的50.98%。(3)由于水热条件及地理条件的差异,植物功能型(不考虑栽培作物)在各区域表现出较大差异,如多年生杂类草草原主要分布于北部地区,在南部并不存在这种植物功能型;森林类型的功能型主要分布于中、南部地区,且结构复杂、类型多样。(4)除栽培作物表现出较好的整体性和连通性,其他植物功能型均表现出不同程度的破碎化和离散化。(5)山西植物功能型整体上表现出较高的多样性,其中中部地区比其他地区的多样性和破碎化程度高,斑块类型更加趋向于离散的小斑块状,北部地区则以一年一熟栽培作物占明显优势,表现出较强的优势度,而南部地区并没有表现出很强的破碎度或优势度。