Rhizobiales-like Phosphatase 2 from Arabidopsis thaliana Is a Novel Phospho-tyrosine-specific Phospho-protein Phosphatase (PPP) Family Protein Phosphatase

Cellular signaling through protein tyrosine phosphorylation is well established in mammalian cells. Although lacking the classic tyrosine kinases present in humans, plants have a tyrosine phospho-proteome that rivals human cells. Here we report a novel plant tyrosine phosphatase from Arabidopsis thaliana (AtRLPH2) that, surprisingly, has the sequence hallmarks of a phospho-serine/threonine phosphatase belonging to the PPP family. Rhizobiales/Rhodobacterales/Rhodospirillaceae-like phosphatases (RLPHs) are conserved in plants and several other eukaryotes, but not in animals. We demonstrate that AtRLPH2 is localized to the plant cell cytosol, is resistant to the classic serine/threonine phosphatase inhibitors okadaic acid and microcystin, but is inhibited by the tyrosine phosphatase inhibitor orthovanadate and is particularly sensitive to inhibition by the adenylates, ATP and ADP. AtRLPH2 displays remarkable selectivity toward tyrosine-phosphorylated peptides versus serine/threonine phospho-peptides and readily dephosphorylates a classic tyrosine phosphatase protein substrate, suggesting that in vivo it is a tyrosine phosphatase. To date, only one other tyrosine phosphatase is known in plants; thus AtRLPH2 represents one of the missing pieces in the plant tyrosine phosphatase repertoire and supports the concept of protein tyrosine phosphorylation as a key regulatory event in plants.     

Rapid purification of bovine kidney branched-chain 2-oxoacid dehydrogenase complex containing endogenous kinase activity

Acid and alkaline phosphatase activity, determined by the hydrolysis of p-nitrophenyl phosphate, was found in preparations of microtubules purified from bovine brain by temperature-dependent assembly-disassembly and ion-exchange chromatography. Phosphocellulose-purified tubulin contained an associated acid phosphatase activity, stimulated by Mg2+ and by Zn2+. Alkaline phosphatase activity with a pH optimum of 10.4 was measured in a fraction of microtubule-associated proteins (MAPs). Kinetics and the effects of sodium fluoride, sodium tartrate, sulfhydryl-blocking agents, EDTA and Zn2+ are reported.     

Reduced expression of kinase-associated phosphatase in cortical dendrites of MAP2-deficient mice

We previously demonstrated that cAMP-dependent protein kinase was reduced in the dendrites of MAP2-deficient mice. In this study, we compared the expression of various protein phosphatases (PPs) between wild-type and map2(-/-) dendrites. Kinase-associated phosphatase (KAP) was the only PP which showed difference between the two phenotypes: (1) the expression of KAP was reduced in map2(-/-) cortical dendrites, and (2) the amount of KAP bound to microtubules was reduced in map2(-/-) brains. We also demonstrated in cultured neuroblastoma cells that KAP is not only expressed in dividing cells, but also in the neurites of differentiated cells. Our findings propose that KAP, which has been reported to function in cell-cycle control, has an as yet uncovered role in regulating dendritic functions. We also propose MAP2-deficient mice as an ideal system for identifying protein phosphatases essential for dendritic functions.     

野生大豆GsPP2CA和GsPKA对GsSnRK1.1蛋白活性的调控

为了明确GsSnRK1.1蛋白激酶在野生大豆生长发育中的具体调控机制,该研究利用酵母二元杂交技术发现了蛋白激酶GsSnRK1.1的互作蛋白GsPP2CA和GsPKA,利用原核表达系统对GsSnRK1.1、GsPP2CA和GsPKA进行了表达和纯化用于Pull down和体外磷酸化分析,并在酵母中研究了GsPP2CA和GsPKA对GsSnRK1.1蛋白活性的调控功能。结果表明：(1)GsSnRK1.1与GsPP2CA和GsPKA具有物理互作关系,Phos Tag和pPKDsub特异性磷酸化抗体检测发现,GsSnRK1.1的Thr176磷酸化可以被蛋白磷酸酶GsPP2CA去磷酸化,GsPKA可能会磷酸化GsSnRK1.1的其他潜在磷酸化位点,进而竞争性抑制GsSnRK1.1的Thr176位点的磷酸化水平。(2)将这些基因回补进入酵母ARY330( snf1/ reg1/ sit4)突变株系中,发现共转化GsSnRK1.1和GsPP2CA或GsPKA的转化子可在非葡萄糖碳源和高葡萄糖碳源的选择培养基上正常生长,GsPP2CA、GsPKA可以替代Reg1和Sit4降低GsSnRK1.1过度磷酸化对酵母细胞产生的毒害作用,进而调控GsSnRK1.1对非发酵型碳源的利用。     

Regulated activity of PP2A–B55δ is crucial for controlling entry into and exit from mitosis in Xenopus egg extracts

Entry into mitosis depends on the activity of cyclin‐dependent kinases (CDKs). Conversely, exit from mitosis occurs when mitotic cyclins are degraded, thereby extinguishing CDK activity. Exit from mitosis must also require mitotic phosphoproteins to revert to their interphase hypophosphorylated forms, but there is a controversy about which phosphatase(s) is/are responsible for dephosphorylating the CDK substrates. We find that PP2A associated with a B55δ subunit is relatively specific for a model mitotic CDK substrate in Xenopus egg extracts. The phosphatase activity measured by this substrate is regulated during the cell cycle—high in interphase and suppressed during mitosis. Depletion of PP2A–B55δ (in interphase) from ‘cycling’ frog egg extracts accelerated their entry into mitosis and kept them indefinitely in mitosis. When PP2A–B55δ was depleted from mitotic extracts, however, exit from mitosis was hardly delayed, showing that other phosphatase(s) are also required for mitotic exit. Increasing the concentration of PP2A–B55δ in extracts by adding recombinant enzyme inhibited the entry into mitosis. This form of PP2A seems to be a key regulator of entry into and exit from mitosis.     

Role of protein phosphatase 2C from bovine adrenal chromaffin cells in the dephosphorylation of phospho-serine 40 tyrosine hydroxylase

Tyrosine hydroxylase (TH) is the rate-limiting enzyme in the synthesis of catecholamines. It is dephosphorylated by protein phosphatase (PP) 2A and PP2C. In this study we used a fixed amount of bacterially expressed rat TH (5 microM), phosphorylated only at serine 40 (pSer40TH), to determine the PP activities against this site that are present in extracts from the bovine adrenal cortex, adrenal medulla, adrenal chromaffin cells and rat striatum. We found that PP2C was the main TH phosphatase activity in extracts from the adrenal medulla and adrenal chromaffin cells. In adrenal cortex extracts PP2C and PP2A activities toward pSer40TH did not differ significantly. PP2A was the main TH phosphatase activity in extracts from rat striatum. Kinetic studies with extracts from adrenal chromaffin cells showed that when higher concentrations of pSer40TH (> 5 microM) were used the activity of PP2C increased more than the activity of PP2A. PP2C was maximally activated by 1.25 mM Mn2+ and by 5 mM Mg2+ but was inhibited by calcium. Our data suggest a more important role for PP2C than was previously suggested in the dephosphorylation of serine 40 on TH.     

Phosphorylation of protein Phosphatase-1 isoforms by cdc2-cyclin B in vitro

Protein Phosphatase-1 is phosphorylated in vitro by cdc2-cyclin B (Villa-Moruzzi, FEBS Lett 304: 211-215, 1992). In the present study we show that all the three Phosphatase-1 isoforms, , 1, , are phosphorylated by cdc2-cyclin B. Phosphorylation is specific for this kinase and involves a C-terminal Thr. This site is most likely Thr 320 in a (shown by others to be phosphorylated also by cdc2-cyclin A). Such Thr is conserved in 1, and in the testis-specific 2, and is the only Thr that fits the cdc2-consensus sequence in the C-terminal region. Phosphorylation of Phosphatase-1 purified from skeletal muscle, which is a mixture of the a, 1 and isoforms, is up to 0.4 mol/mol and induces 30-35% enzyme inactivation. Following tryptic proteolysis each isoform yields a distinct phosphopeptide map. This is in agreement with the different sequences of the isoforms in the C-terminal regions and may be useful to distinguish the isoforms lts suggest that all the Phosphatase-1 isoforms may be potentially regulated at M-phase.     

Structure and substrate recognition of the Staphylococcus aureus protein tyrosine phosphatase PtpA

Phosphosignaling through pSer/pThr/pTyr is emerging as a common signaling mechanism in prokaryotes. The human pathogen Staphylococcus aureus produces two low-molecular-weight protein tyrosine phosphatases (PTPs), PtpA and PtpB, with unknown functions. To provide the structural context for understanding PtpA function and substrate recognition, establish PtpA's structural relations within the PTP family, and provide a framework for the design of specific inhibitors, we solved the crystal structure of PtpA at 1 Å resolution. While PtpA adopts the common, conserved PTP fold and shows close overall similarity to eukaryotic PTPs, several features in the active site and surface organization are unique and can be explored to design selective inhibitors. A peptide bound in the active site mimics a phosphotyrosine substrate, affords insight into substrate recognition, and provides a testable substrate prediction. Genetic deletion of ptpA or ptpB does not affect in vitro growth or cell wall integrity, raising the possibility that PtpA and PtpB have specialized functions during infection.     

Porcine liver low Mr phosphotyrosine protein phosphatase: The amino acid sequence

Porcine low M_r phosphotyrosine protein phosphatase has been purified and the complete amino acid sequence has been determined. Both enzymic and chemical cleavages are used to obtain protein fragments. FAB mass spectrometry and enzymic subdigestion followed by Edman degradation have been used to determine the structure of the NH₂-terminal acylated tryptic peptide. The enzyme consists of 157 amino acid residues, is acetylated at the NH₂-terminus, and has arginine as COOH-terminal residue. It shows kinetic parameters very similar to other known low Mr PTPases. This PTPase is strongly inhibited by pyridoxal 5-phosphate (K=21M) like the low Mr PTPases from bovine liver, rat liver (AcP2 isoenzyme), and human erythrocyte (Bslow isoenzyme). The comparison of the 40–73 sequence with the corresponding sequence of other low Mr PTPases from different sources demonstrates that this isoform is highly homologous to the isoforms mentioned above, and shows a lower homology degree with respect to rat AcP1 and human Bfast isoforms. A classification of low M_r PTPase isoforms based on the type-specific sequence and on the sensitivity to pyridoxal 5-phosphate inhibition has been proposed.Abbreviations used PTPase phosphotyrosine protein phosphatase - TFA trifluoroacetic acid - SDS sodium dodecylsulfate - T tryptic peptides - SP endoproteinase Glu-C peptides - FAB fast atom bombardment - Ac acetyl - HPLC high-performance liquid chromatography - OPA o-phtaldialdehyde - PMSF phenylmethylsulfonyl fluoride - CD45 leukocyte common-antigen PTPase - LAR leukocyte-antigen-related PTPase - PTP IB human placental PTPase     

不同磷源对金龟子绿僵菌CQMa102生物量及生成胞外磷酸酶的影响(英文)

金龟子绿僵菌Metarhizium anisopliae是一种广泛分布于世界各地土壤中的重要的昆虫病原真菌。已有研究表明,胞外磷酸酶在绿僵菌侵染并致死寄主过程中发挥了重要作用。利用摇瓶培养方法探究了无机磷(KH2PO4)、简单有机磷(植酸钠、磷酸苯二钠)和蛋白有机磷(酪蛋白)为单独磷源条件下,绿僵菌生物量、产胞外酸性磷酸酶以及酪氨酸蛋白磷酸酶、丝/苏氨酸蛋白磷酸酶活性的变化。实验结果显示:加入酪蛋白的培养基,最有利于绿僵菌生长、胞外蛋白的分泌和产酸性磷酸酶;其次为加入KH2PO4和磷酸苯二钠的培养基;加入植酸钠的培养基不利于绿僵菌的生长代谢。然而,加入磷酸苯二钠的培养基,最有利于酪氨酸蛋白磷酸酶、丝/苏氨酸蛋白磷酸酶的分离纯化。     

Screening and characterization of microbial inhibitors against eukaryotic protein phosphatases (PP1 and PP2A)

AIM: To identify novel microbial inhibitors of protein phosphatase 1 (PP1). METHODS AND RESULTS: 750 actinomycetes and 408 microfungi were isolated from Sabah forest soils and screened for production of potential PP1 inhibitors using an in vivo screening system, in which candidate inhibitors were identified through mimicking the properties of PP1-deficient yeast cells. Acetone extracts of two fungi, H9318 (Penicillium) and H9978 (non-Penicillium) identified in this way showed inhibitory activity towards both mammalian PP1 and PP2A in an in vitro phosphatase assay, while extract from H7520 (Streptomyces) inhibited PP2A but not PP1. Consistently, using a drug-induced haploinsufficiency test, strains with either reduced PP1 or PP2A function were hypersensitive to H9318 and H9978 extracts whereas only the latter strain showed hypersensitivity to H7250 extract. H9318 extract was fractionated using RP-HPLC into two active peaks (S1 and S2). A yeast strain with reduced PP1 function showed hypersensitivity to fraction S2 whereas a strain with reduced PP2A function was hypersensitive to fraction S1. However, S1 and S2 inhibited both PP1 and PP2A activities to a similar extent. CONCLUSION: Three candidate PP inhibitors have been identified. SIGNIFICANCE AND IMPACT OF THE STUDY: Further development may generate useful research tools and ultimately therapeutic agents.     

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.     

Rat liver lowMr phosphotyrosine protein phosphatase isoenzymes: Purification and amino acid sequences

Two lowM _r phosphotyrosine protein phosphatases have been isolated from rat liver. The enzymes were previously known as lowM _r acid phosphatases, but several recent studies have demonstrated that this family of enzymes possesses specific phosphotyrosine protein phosphatase activity. We determined the complete amino acid sequences of the two isoenzymes and named them AcP1 and AcP2. Both consist of 157 amino acid residues, are acetylated at the NH₂-terminus, and have His as the COOH-terminus. The molecular weights calculated from the sequences are 18,062 for AcP1 and 17,848 for AcP2. They are homologous except in the 40–73 zone, where about 50% of residues are different. This fact suggests that the two isoenzymes are produced by an alternative splicing mechanism. There is no homology between these two isoenzymes and the receptor-like phosphotyrosine protein phosphatases LAR, CD45, human placenta PTPase 1B, and rat brain PTPase-1. AcP1 and AcP2 are also distinct from rat liver PTPase-1 and PTPase-2, since these last enzymes have higher molecular weights. AcP1 differs from AcP2 with respect to (1) substrate affinity and (2) its sensitivity to activators and inhibitors, thus suggesting a their different physiological function.     

Inhibition of Ca2+/calmodulin-dependent phosphatase activity by regucalcin in rat liver cytosol: Involvement of calmodulin binding

The regulatory effect of regucalcin on Ca²⁺/calmodulin-dependent phosphatase activity and the binding of regucalcin to calmodulin was investigated. Phosphatase activity toward phosphotyrosine, phosphoserine, and phosphothreonine in rat liver cytosol was significantly increased by the addition of Ca²⁺ (100 μM) and calmodulin (0.30 μM). Thess increases were clearly inhibited by the addition of regucalcin (0.50–1.0 μM) into the enzyme reaction mixture. The cytosolic phosphoamino acid phosphatase activity was significantly elevated by the presence of anti-regucalcin monoclonal antibody (0.2 μg/ml), suggesting that endogenous regucalcin in the cytosol has an inhibitory effect on the enzyme activity. This elevation was prevented by the addition of regucalcin (0.50 μM). Purified calcineurin phosphatase activity was significantly increased by the addition of calmodulin (0.12 μM) in the presence of Ca²⁺ (1 and 10 μM). This increase was completely inhibited by the presence of regucalcin (0.12 μM). The inhibitory effect of regucalcin was reversed by the addition of calmodulin with the higher concentration (0.36 μM). Regucalcin has been demonstrated to bind on calmodulin-agarose beads by analysis with sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The present study demonstrates that regucalcin inhibits Ca²⁺/calmodulin-dependent protein phosphatase activity in rat liver cytosol, and that regucalcin can bind to calmodulin. J. Cell. Biochem. 71:140–148, 1998. © 1998 Wiley-Liss, Inc.     

Regulation of aflatoxin production by Ca(2+)/calmodulin-dependent protein phosphorylation and dephosphorylation

To elucidate Ca(2+)-mediated regulation of aflatoxin production, the status of Ca(2+)/calmodulin-dependent protein phosphorylation and dephosphorylation was investigated employing toxigenic and non-toxigenic strains of Aspergillus parasiticus. Incubation of cytoplasmic extracts with [gamma-(32)P]ATP followed by SDS-PAGE and autoradiography revealed total absence of protein phosphorylation during periods corresponding to aflatoxin production in the toxigenic strain (NRRL 2999). In contrast, protein phosphorylation was unaffected in the non-toxigenic strain (SRRC 255). Aflatoxin production in the toxigenic strain was also accompanied by enhanced (26-fold) activity of calcineurin (calmodulin-dependent protein phosphatase 2B) concomitant with a lowered (6-fold) activity of calmodulin-dependent protein kinase. In addition, the in vitro activity of Ca(2+)/calmodulin-dependent protein kinase was susceptible to dose-dependent inhibition by aflatoxin. Since calcineurin remains active in the absence of phosphorylation by calmodulin-dependent protein kinase, it is suggested that calcineurin-mediated dephosphorylation of regulatory enzymes ensures continued production of aflatoxins.     

MicroRNA-141 promotes the proliferation of non-small cell lung cancer cells by regulating expression of PHLPP1 and PHLPP2

The dysregulation of microRNAs (miRNAs) is crucially implicated in the development of various cancers. In this study, we explored the biological role of miR-141 in non-small cell lung cancer (NSCLC). miR-141 expression was significantly up-regulated in NSCLC tissues, and its overexpression accelerated NSCLC cell proliferation in vitro and tumor growth in vivo. We subsequently identified the antagonists of PI3K/AKT signaling, PH domain leucine-rich-repeats protein phosphatase 1 (PHLPP1) and PHLPP2, as direct targets of miR-141. Re-introduction of PHLPP1 and PHLPP2 abrogated miR-141-induced proliferation of NSCLC cells. Together, the results of this study suggest that miR-141 and its targets PHLPP1 and PHLPP2 play critical roles in NSCLC tumorigenesis, and provide potential therapeutic targets for NSCLC treatment.     

Structural basis of interaction between protein tyrosine phosphatase PCP-2 and β-catenin

PCP-2 is a member of receptor-like protein tyrosine phosphatase of the MAM domain family. To investigate which part of PCP-2 was involved in its interaction with β-catenin, we constructed various deletion mutants of PCP-2. These PCP-2 mutants and wild-type PCP-2 were co-transfected into BHK-21 cells with β-catenin individually. Anin vivo binding assay revealed that the expression of wild-type PCP-2, PCP-2 ΔC1C2 (deleted PCP-2 without both PTP domains) and PCP-2 ΔC2 (deleted PCP-2 without the second PTP domain) could be immunoprecipitated by anti-catenin antibody in every co-transfection, but PCP-2 EXT (deleted PCP-2 without the juxtamembrane region and both PTP domains) was missing, which implied that PCP-2 and β-catenin could associate directly and the juxtamembrane region in PCP-2 was sufficient for the process.     

Developmental Regulation of Protein Phosphatase Types 1 and 2A in Post-Hatch Chicken Brain

The activity and subcellular distribution of protein phosphatases 1 and 2A were measured in chicken forebrain and cerebellum during post-hatch development. At all post-hatch ages, a large proportion of PP1 and PP2A was membrane bound and these enzymes were less active than their cytosolic counterparts. The protein concentration of PP1 in the membranes increased 40% between 2 and 14 days and a further 60% between 14 days and adult, whereas the PP1 enzyme activity in the membranes progressively decreased. In contrast to PP1, the protein concentration of PP2A remained constant in all fractions during post-hatch development, and the enzyme activity of PP2A did not change except for a decrease in the membrane-bound activity between 2 and 14 days. These results show that the subcellular distribution and activity of PP1 is selectively regulated during post-hatch development and that membrane association and inactivation of PP1 are independent events.     

The RING finger E3 ligases PIR1 and PIR2 mediate PP2CA degradation to enhance abscisic acid response in Arabidopsis

Recent work has established a core ABA signaling pathway in which A‐type PP2C protein phosphatases act as central negative modulators. Although ABA signaling inhibits PP2C activity through ABA‐receptor complex, it remains unknown if other mechanisms exist to modulate the level of PP2Cs. Here, we identified a RING domain ubiquitin E3 ligase, PIR1 (PP2CA interacting RING finger protein 1), that interacted with PP2CA. Of the two splicing isoforms, PIR1.2 was isolated from leaf tissue. The PIR1.2 exhibited E3 ligase activity and determined PP2CA stability in the presence of ABA. Consistent with the conclusion that PIR1 promotes ABA signaling by removing PP2CA, a negative modulator, the pir1 knockout mutant displayed an ABA‐hyposensitive phenotype. We further showed that PIR2, the closest homologue of PIR1.2, also interacted with PP2CA. Although the pir2 knockout mutant did not display altered ABA response, the pir1‐1/pir2 double mutant became more insensitive to ABA than the wild‐type or pir1‐1 and pir2 single mutants. Using a cell‐free degradation assay, ABA promoted degradation of PP2CA, however, such degradation was delayed when incubated with protein extract prepared from the pir1‐1/pir2 double mutant. Our data suggest that PIR1 and PIR2 positively modulate ABA signaling by targeting PP2CA for degradation.