Missense substitutions reflecting regulatory control of transmitter phosphatase activity in two‐component signalling

Negative control in two‐component signal transduction results from sensor transmitter phosphatase activity for phospho‐receiver dephosphorylation. A hypothetical mechanism for this reaction involves a catalytic residue in the H‐box active‐site region. However, a complete understanding of transmitter phosphatase regulation is hampered by the abundance of kinase‐competent, phosphatase‐defective missense substitutions (K⁺ P^– phenotype) outside of the active‐site region. For the Escherichia coli NarX sensor, a model for the HisKA_3 sequence family, DHp domain K⁺ P^– mutants defined two classes. Interaction mutants mapped to the active site‐distal base of the DHp helix 1, whereas conformation mutants were affected in the X‐box region of helix 2. Thus, different types of perturbations can influence transmitter phosphatase activity indirectly. By comparison, K⁺ P^– substitutions in the HisKA sensors EnvZ and NtrB additionally map to a third region, at the active site‐proximal top of the DHp helix 1, independently identified as important for DHp‐CA domain interaction in this sensor class. Moreover, the NarX transmitter phosphatase activity was independent of nucleotides, in contrast to the activity for many HisKA family sensors. Therefore, distinctions involving both the DHp and the CA domains suggest functional diversity in the regulation of HisKA and HisKA_3 transmitter phosphatase activities.     

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。     

Occurrence of the ACP 1 0 allele in Czechoslovakia

Summary During a paternity test an unexpected type of red cell acid phosphatase isozyme (ACP₁) was found in one family. The mother was of type A and type B was diagnosed in the son. The whole family was then subjected to ACP₁ phenotyping and to the enzyme assay. Five members of the family were found to have unexpected types of ACP₁ isozymes. The average activity was approx. 50% of normal values. It is presumed that a silent ACP ₁ ⁰ allele was found in the family investigated and that the grandfather was its first carrier.     

Molecular cloning and biochemical characterization of bovine retina calcineurin

Calcineurin (CaN) is a member of ser/thr protein phosphatase family. Earlier, we have reported that CaN is present in all eye tissues, although the activity and protein expression varied (Seitz et al., Invest Opthalmol Vis Sci, 43:15–21, 2002). We have isolated a full-length cDNA encoding bovine retina CaN. The CaN A subunit consists of 511 amino acid residues. A 10 amino acid (ATVEAIEADE) deletion before the autoinhibitory domain was observed in bovine retina CaN A compared to bovine brain CaN A. The study on CaN activity and regulation demonstrated that different metal ions have different effects on its phosphatase activity. Ni²⁺ was found to be the strongest stimulator, while Zn²⁺ was found to inhibit CaN phosphatase activity. Mn²⁺ was a relatively less effective stimulator compared to Ni²⁺. Fe²⁺ was also able to stimulate CaN phosphatase activity; in contrast, a previous study found Fe²⁺ slightly inhibited CaN activity from bovine brain. The residues at 97–201 were found to be essential for bovine retina CaN A phosphatase activity. The residues at 407–456 also had an inhibitory effect on CaN A phosphatase activity in addition to the previously known autoinhibitory domain at 457–480. These observations suggest that bovine retina CaN A might possess some distinct structural characteristics.     

Substrate-dependent metal preference of PPM1H, a cancer-associated protein phosphatase 2C: comparison with other family members

Protein phosphatase 2C (PP2C) family is characterized by requirement of metal cation for phosphatase activity. We previously established that PPM1H is a cancer-associated member of the PP2C family. Here we further characterized the phosphatase activity of PPM1H, focusing on its dependence on metal cation. PPM1H possesses the potential to dephosphorylate p-nitrophenyl phosphate (pNPP), casein and phosphopeptides. Interestingly, PPM1H shows the metal preference that is varied depending on the substrate (substrate-dependent metal preference); PPM1H prefers Mn²⁺ when pNPP or phosphopeptides is used as a substrate. Meanwhile, a preference for Mg²⁺ is displayed by PPM1H with casein as a substrate. When both cations are added to the reaction, the degree of the effect is always closer to that with Mn²⁺ alone, irrespective of the substrate. This preponderance of Mn²⁺ is explained by its greater affinity for PPM1H than Mg²⁺. From the literature the substrate-dependent metal preference appears to be shared by other PP2Cs. According to the crystal structure, a binuclear metal center of PP2C plays an important role for coordinating the substrate and nucleophilic waters in the active site. Therefore, the differences in the size, preferred geometry and coordination requirements between two metals, in relation to the substrate, may be responsible for this intriguing property.     

Phosphorylated TandeMBP: A unique protein substrate for protein phosphatase assay

To analyze a variety of protein phosphatases, we developed phosphorylated TandeMBP (P-TandeMBP), in which two different mouse myelin basic protein isoforms were fused in tandem, as a protein phosphatase substrate. P-TandeMBP was prepared efficiently in four steps: (1) phosphorylation of TandeMBP by a protein kinase mixture (Ca²⁺/calmodulin-dependent protein kinase Iδ, casein kinase 1δ, and extracellular signal-regulated kinase 2); (2) precipitation of both P-TandeMBP and protein kinases to remove ATP, Pi, and ADP; (3) acid extraction of P-TandeMBP with HCl to remove protein kinases; and (4) neutralization of the solution that contains P-TandeMBP with Tris. In combination with the malachite green assay, P-TandeMBP can be used to detect protein phosphatase activity without using radioactive materials. Moreover, P-TandeMBP served as an efficient substrate for PPM family phosphatases (PPM1A, PPM1B, PPM1D, PPM1F, PPM1G, PPM1H, PPM1K, and PPM1M) and PPP family phosphatase PP5. Various phosphatase activities were also detected with high sensitivity in gel filtration fractions from mouse brain using P-TandeMBP. These results indicate that P-TandeMBP might be a powerful tool for the detection of protein phosphatase activities.     

Ppm1E is an in cellulo AMP-activated protein kinase phosphatase

Activation of 5′-AMP-activated protein kinase (AMPK) is believed to be the mechanism by which the pharmaceuticals, metformin and phenformin, exert their beneficial effects for treatment of type 2 diabetes. These biguanide drugs elevate 5′-AMP, which allosterically activates AMPK and promotes phosphorylation on Thr172 of AMPK catalytic α subunits. Although kinases phosphorylating this site have been identified, phosphatases that dephosphorylate it are unknown. The aim of this study is to identify protein phosphatase(s) that dephosphorylate AMPKα-Thr172 within cells. Our initial data indicated that members of the protein phosphatase ce:sup>/ce:sup>/Mn²⁺-dependent (PPM) family and not those of the PPP family of protein serine/threonine phosphatases may be directly or indirectly inhibited by phenformin. Using antibodies raised to individual Ppm phosphatases that facilitated the assessment of their activities, phenformin stimulation of cells was found to decrease the ce:sup>/ce:sup>/Mn²⁺-dependent protein serine/threonine phosphatase activity of Ppm1E and Ppm1F, but not that attributable to other PPM family members, including Ppm1A/PP2Cα. Depletion of Ppm1E, but not Ppm1A, using lentiviral-mediated stable gene silencing, increased AMPKα-Thr172 phosphorylation approximately three fold in HEK293 cells. In addition, incubation of cells with low concentrations of phenformin and depletion of Ppm1E increased AMPK phosphorylation synergistically. Ppm1E and the closely related Ppm1F interact weakly with AMPK and assays with lysates of cells stably depleted of Ppm1F suggests that this phosphatase contributes to dephosphorylation of AMPK. The data indicate that Ppm1E and probably PpM1F are in cellulo AMPK phosphatases and that Ppm1E is a potential anti-diabetic drug target.     

Evolutionary and Structural Analyses of Mammalian Haloacid Dehalogenase-type Phosphatases AUM and Chronophin Provide Insight into the Basis of Their Different Substrate Specificities

Mammalian haloacid dehalogenase (HAD)-type phosphatases are an emerging family of phosphatases with important functions in physiology and disease, yet little is known about the basis of their substrate specificity. Here, we characterize a previously unexplored HAD family member (gene annotation, phosphoglycolate phosphatase), which we termed AUM, for aspartate-based, ubiquitous, Mg²⁺-dependent phosphatase. AUM is a tyrosine-specific paralog of the serine/threonine-specific protein and pyridoxal 5′-phosphate-directed HAD phosphatase chronophin. Comparative evolutionary and biochemical analyses reveal that a single, differently conserved residue in the cap domain of either AUM or chronophin is crucial for phosphatase specificity. We have solved the x-ray crystal structure of the AUM cap fused to the catalytic core of chronophin to 2.65 Å resolution and present a detailed view of the catalytic clefts of AUM and chronophin that explains their substrate preferences. Our findings identify a small number of cap domain residues that encode the different substrate specificities of AUM and chronophin.     

V‐ATPase deactivation in blowfly salivary glands is mediated by protein phosphatase 2C

The activity of vacuolar H⁺‐ATPase (V‐ATPase) in the apical membrane of blowfly (Calliphora vicina) salivary glands is regulated by the neurohormone serotonin (5‐HT). 5‐HT induces, via protein kinase A, the phosphorylation of V‐ATPase subunit C and the assembly of V‐ATPase holoenzymes. The protein phosphatase responsible for the dephosphorylation of subunit C and V‐ATPase inactivation is not as yet known. We show here that inhibitors of protein phosphatases PP1 and PP2A (tautomycin, ocadaic acid) and PP2B (cyclosporin A, FK‐506) do not prevent V‐ATPase deactivation and dephosphorylation of subunit C. A decrease in the intracellular Mg²⁺ level caused by loading secretory cells with EDTA‐AM leads to the activation of proton pumping in the absence of 5‐HT, prolongs the 5‐HT‐induced response in proton pumping, and inhibits the dephosphorylation of subunit C. Thus, the deactivation of V‐ATPase is most probably mediated by a protein phosphatase that is insensitive to okadaic acid and that requires Mg²⁺, namely, a member of the PP2C protein family. By molecular biological techniques, we demonstrate the expression of at least two PP2C protein family members in blowfly salivary glands. © 2009 Wiley Periodicals, Inc.     

The K+- and HCO3−-stimulated phosphatase activities in renal microsomes of rats

The K⁺-stimulated phosphatase activity of microsomes from rat kidney was not inhibited by l-phenylalanine, but the HCO₃^?-stimulated phosphatase activity was markedly inhibited by l-phenylalanine. Valinomycin enhanced the HCO₃^?-stimulated phosphatase activity, but did not enhance the K⁺-stimulated phosphatase activity. Ouabain did not inhibit the HCO₃^?-stimulated phosphatase activity, but inhibited the K⁺-stimulated phosphatase activity.The renal K⁺-stimulated phosphatase activity was suppressed to 40% of the control values by adrenalectomy, but the renal HCO₃^?-stimulated phosphatase activity was little suppressed by adrenalectomy. The renal K⁺-stimulated phosphatase activity in intact and adrenalectomized rats was found to be significantly elevated, in a manner similar to the elevation of the renal (Na⁺ + K⁺)-ATPase activity by aldosterone treatment (P < 0.02).     

Direct Determination of Phosphatase Activity from Physiological Substrates in Cells

A direct and continuous approach to determine simultaneously protein and phosphate concentrations in cells and kinetics of phosphate release from physiological substrates by cells without any labeling has been developed. Among the enzymes having a phosphatase activity, tissue non-specific alkaline phosphatase (TNAP) performs indispensable, multiple functions in humans. It is expressed in numerous tissues with high levels detected in bones, liver and neurons. It is absolutely required for bone mineralization and also necessary for neurotransmitter synthesis. We provided the proof of concept that infrared spectroscopy is a reliable assay to determine a phosphatase activity in the osteoblasts. For the first time, an overall specific phosphatase activity in cells was determined in a single step by measuring simultaneously protein and substrate concentrations. We found specific activities in osteoblast like cells amounting to 116 ± 13 nmol min^-1 mg^-1 for PPi, to 56 ± 11 nmol min^-1 mg^-1 for AMP, to 79 ± 23 nmol min^-1 mg^-1 for beta-glycerophosphate and to 73 ± 15 nmol min^-1 mg^-1 for 1-alpha-D glucose phosphate. The assay was also effective to monitor phosphatase activity in primary osteoblasts and in matrix vesicles. The use of levamisole – a TNAP inhibitor- served to demonstrate that a part of the phosphatase activity originated from this enzyme. An IC₅₀ value of 1.16 ± 0.03 mM was obtained for the inhibition of phosphatase activity of levamisole in osteoblast like cells. The infrared assay could be extended to determine any type of phosphatase activity in other cells. It may serve as a metabolomic tool to monitor an overall phosphatase activity including acid phosphatases or other related enzymes.     

The myosin phosphatase targeting protein (MYPT) family: a regulated mechanism for achieving substrate specificity of the catalytic subunit of protein phosphatase type 1δ

The mammalian MYPT family consists of the products of five genes, denoted MYPT1, MYPT2, MBS85, MYPT3 and TIMAP, which function as targeting and regulatory subunits to confer substrate specificity and subcellular localization on the catalytic subunit of type 1δ protein serine/threonine phosphatase (PP1cδ). Family members share several conserved domains, including an RVxF motif for PP1c binding and several ankyrin repeats that mediate protein–protein interactions. MYPT1, MYPT2 and MBS85 contain C-terminal leucine zipper domains involved in dimerization and protein–protein interaction, whereas MYPT3 and TIMAP are targeted to membranes via a C-terminal prenylation site. All family members are regulated by phosphorylation at multiple sites by various protein kinases; for example, Rho-associated kinase phosphorylates MYPT1, MYPT2 and MBS85, resulting in inhibition of phosphatase activity and Ca²⁺ sensitization of smooth muscle contraction. A great deal is known about MYPT1, the myosin targeting subunit of myosin light chain phosphatase, in terms of its role in the regulation of smooth muscle contraction and, to a lesser extent, non-muscle motile processes. MYPT2 appears to be the key myosin targeting subunit of myosin light chain phosphatase in cardiac and skeletal muscles. MBS85 most closely resembles MYPT2, but little is known about its physiological function. Little is also known about the physiological role of MYPT3, although it is likely to target myosin light chain phosphatase to membranes and thereby achieve specificity for substrates involved in regulation of the actin cytoskeleton. MYPT3 is regulated by phosphorylation by cAMP-dependent protein kinase. TIMAP appears to target PP1cδ to the plasma membrane of endothelial cells where it serves to dephosphorylate proteins involved in regulation of the actin cytoskeleton and thereby control endothelial barrier function. With such a wide range of regulatory targets, MYPT family members have been implicated in diverse pathological events, including hypertension, Parkinson’s disease and cancer.     

A Unique Protein Phosphatase with Kelch-Like Domains (PPKL) in Plasmodium Modulates Ookinete Differentiation,Motility and Invasion

Protein phosphorylation and dephosphorylation (catalysed by kinases and phosphatases, respectively) are post-translational modifications that play key roles in many eukaryotic signalling pathways, and are often deregulated in a number of pathological conditions in humans. In the malaria parasite Plasmodium, functional insights into its kinome have only recently been achieved, with over half being essential for blood stage development and another 14 kinases being essential for sexual development and mosquito transmission. However, functions for any of the plasmodial protein phosphatases are unknown. Here, we use reverse genetics in the rodent malaria model, Plasmodium berghei, to examine the role of a unique protein phosphatase containing kelch-like domains (termed PPKL) from a family related to Arabidopsis BSU1. Phylogenetic analysis confirmed that the family of BSU1-like proteins including PPKL is encoded in the genomes of land plants, green algae and alveolates, but not in other eukaryotic lineages. Furthermore, PPKL was observed in a distinct family, separate to the most closely-related phosphatase family, PP1. In our genetic approach, C-terminal GFP fusion with PPKL showed an active protein phosphatase preferentially expressed in female gametocytes and ookinetes. Deletion of the endogenous ppkl gene caused abnormal ookinete development and differentiation, and dissociated apical microtubules from the inner-membrane complex, generating an immotile phenotype and failure to invade the mosquito mid-gut epithelium. These observations were substantiated by changes in localisation of cytoskeletal tubulin and actin, and the micronemal protein CTRP in the knockout mutant as assessed by indirect immunofluorescence. Finally, increased mRNA expression of dozi, a RNA helicase vital to zygote development was observed in ppkl⁻ mutants, with global phosphorylation studies of ookinete differentiation from 1.5–24 h post-fertilisation indicating major changes in the first hours of zygote development. Our work demonstrates a stage-specific essentiality of the unique PPKL enzyme, which modulates parasite differentiation, motility and transmission.     

Cloning, Sequencing and Characterization of a Novel Phosphatase Gene, phoI, from Soil Bacterium Enterobacter sp. 4

A gene, phoI, coding for a phosphatase from Enterobacter sp. 4 was cloned in Escherichia coli and sequenced. Analysis of the sequence revealed one open reading frame (ORF) that encodes a 269–amino acid protein with a calculated molecular mass of 29 kDa. PhoI belongs to family B acid phosphatase and exhibits 49.4% identity and 62.4% homology to the hel gene from Heamophilus influenzae, which encoded an outer membrane protein (P4). The optimum pH and temperature for phosphatase activity were pH 5.5 and 40°C, respectively. Its specific activity on ρ-nitrophenyl phosphatate was 70 U/mg at pH 5.5 and 40°C. Enzyme activity was inhibited by Al³⁺, EDTA, and DTT, but fivefold activated by Cu²⁺ ion (350 U/mg). PhoI showed a strong synergistic effect when used with a purified E. coli phytase, AppA, to estimate combination effects. Seung Ha Kang and Kwang Keun Cho contributed equally to this work.     

Identification of pseudo ‘phosphothreonyl-specific’ protein phosphatase T with a fraction of polycation-stimulated protein phosphatase 2A

Protein phosphatase T from rat liver, so termed due to its activity toward [³²P-Thr]casein and its marked preference for the phosphopeptide Arg-Arg-Ala-Thr(P)-Val-Ala over its phosphoseryl derivative (Donella Deana, A., Marchiori, F., Meggio, F. and Pinna, L.A. (1982) J. Biol. Chem. 257, 8565–8568), is shown here to belong to the family of type 2A protein phosphatase according to Cohen's nomenclature (Ingebritsen, T.S. and Cohen, P. (1983) Eur. J. Biochem. 132, 255–261). In particular, protein phosphatase T is endowed with phosphorylase phosphatase activity that is stimulated by protamine, histone H1 and heparin, it is inhibited by spermine, it does not bind to heparin-Sepharose and it readily dephosphorylates the phosphopeptide Arg-Arg-Leu-Ser(P)-Ile-Ser-Thr-Glu-Ser reproducing the phosphorylation site of the α-subunit of phosphorylase kinase. The M_r of protein phosphatase T determined by gel filtration under non-denaturating conditions is about 150 kDa and its activity ratio toward histone H1 phosphorylated by protein kinase C versus histone H1 phosphorylated by cAMP-dependent protein kinase is unusually high. Some properties of protein phosphatase T, such as its weak binding to DEAE-cellulose and its high stimulation by protamine as compared to a relatively poor stimulation by histone H1, suggest that it may be similar to subtype 2A_o of protein phosphatase 2A.     

Vertical Profile of Phosphatase Activity in the Sundarban Mangrove Forest,North East Coast of Bay of Bengal,India

Impact of phosphate solubilizing bacteria along with soil phosphatase activity on phosphorous cycle was found to be quiet interesting in the Sundarban mangrove ecosystem. Soil phosphatase activity showed a decreasing pattern with increase in depth [soil phosphatase activity (μg pnp produced g^?1 dry wt of soil) = 906.85 – 5.6316 Depth (cm)] from the deep forest region of the Sundarban forest ecosystem. Soil salinity showed a very little effect on soil phosphatase activity whereas soil temperature and pH was found to show significant impact on the soil phosphatase activity. This ensured that the microbes associated with phosphate mineralization present in the Sundarban forest ecosystem are more tolerant to fluctuation in salinity than that of temperature and pH. A direct correlation was perceptible between the number of phosphate solubilizing bacteria and phosphatase activity in the soil during the study period from 2007 to 2012. Soil phosphate concentration was found to be directly governed by the soil phosphatase activity [The regression equation is: avg PO₄^?3-P (μg g^?1 dry wt of soil) = 0.0311 + 0.000606 soil phosphatase activity (μg pnp produced g^?1 dry wt of soil); R² = 63.2%, p < 0.001, n = 62].     

Crystal Structure of the Bacillus subtilis Phosphodiesterase PhoD Reveals an Iron and Calcium-containing Active Site

The PhoD family of extra-cytoplasmic phosphodiesterases are among the most commonly occurring bacterial phosphatases. The exemplars for this family are the PhoD protein of Bacillus subtilis and the phospholipase D of Streptomyces chromofuscus. We present the crystal structure of B. subtilis PhoD. PhoD is most closely related to purple acid phosphatases (PAPs) with both types of enzyme containing a tyrosinate-ligated Fe³⁺ ion. However, the PhoD active site diverges from that found in PAPs and uses two Ca²⁺ ions instead of the single extra Fe²⁺, Mn²⁺, or Zn²⁺ ion present in PAPs. The PhoD crystals contain a phosphate molecule that coordinates all three active site metal ions and that is proposed to represent a product complex. A C-terminal helix lies over the active site and controls access to the catalytic center. The structure of PhoD defines a new phosphatase active site architecture based on Fe³⁺ and Ca²⁺ ions.     

Alkaline phosphatase activity and the regulation of growth in transformed mammalian cells

The relationship between alkaline phosphatase activity and cell growth has been studied in hamster cells transformed by different carcinogens. About 90% of normal hamster embryo cells were constitutively positive for alkaline phosphatase activity (AP⁺). However, there were no AP⁺ cells in cell lines transformed after treatment with the chemical carcinogens dimethylnitrosamine or 4-nitro-quinoline-N-oxide and 0.02% and 4% AP⁺ cells in cell lines transformed by polyoma virus or Simian virus 40. The glucocorticoid hormone, prednisolone, induced alkaline phosphatase activity in 12% and 44% of the enzyme-negative (AP^?) cells in cell lines transformed by polyoma or Simian virus 40, but this hormone did not induce alkaline phosphatase activity in AP^? cells from cell lines transformed after treatment with the chemical carcinogens. Treatment of polyoma transformed AP^? cells with the mutagen N-methyl-N′-nitro-N-nitro-soguanidine produced AP⁺ cells, whereas no AP⁺ cells were found after mutagen treatment of AP^? cells from the chemically transformed cell lines. Studies on spontaneous segregation in the polyoma transformed cell line has shown that AP⁺ cells segregated AP^? cells both in vitro and in vivo, although no spontaneous segregation was observed from AP^? to AP⁺ cells. AP⁺ cells, compared to AP^? cells, showed a decrease in DNA synthesis, cell multiplication, the ability to form colonies in soft agar and tumorogenicity in animals. AP^? cells induced for alkaline phosphatase activity by prednisolone, showed the same growth properties in vitro as uninduced AP^? cells. The decreased cell growth found in AP⁺ cells which were constitutive for alkaline phosphatase activity was therefore not found in the hormone induced AP^? cells. The results indicate that constitutive alkaline phosphatase activity appears to be related to the regulation of cell growth and that AP^? cells have a selective advantage over AP⁺ cells.