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Journal of Bacteriology Nov 1999Pseudomonas aeruginosa is an opportunistic pathogen that causes infections in eye, urinary tract, burn, and immunocompromised patients. We have cloned and characterized...
Pseudomonas aeruginosa is an opportunistic pathogen that causes infections in eye, urinary tract, burn, and immunocompromised patients. We have cloned and characterized a serine/threonine (Ser/Thr) kinase and its cognate phosphoprotein phosphatase. By using oligonucleotides from the conserved regions of Ser/Thr kinases of mycobacteria, an 800-bp probe was used to screen P. aeruginosa PAO1 genomic library. A 20-kb cosmid clone was isolated, from which a 4.5-kb DNA with two open reading frames (ORFs) were subcloned. ORF1 was shown to encode Ser/Thr phosphatase (Stp1), which belongs to the PP2C family of phosphatases. Overlapping with the stp1 ORF, an ORF encoding Hank's type Ser/Thr kinase was identified. Both ORFs were cloned in pGEX-4T1 and expressed in Escherichia coli. The overexpressed proteins were purified by glutathione-Sepharose 4B affinity chromatography and were biochemically characterized. The Stk1 kinase is 39 kDa and undergoes autophosphorylation and can phosphorylate eukaryotic histone H1. A site-directed Stk1 (K86A) mutant was shown to be incapable of autophosphorylation. A two-dimensional phosphoamino acid analysis of Stk1 revealed strong phosphorylation at a threonine residue and weak phosphorylation at a serine residue. The Stp1 phosphatase is 27 kDa and is an Mn(2+)-, but not a Ca(2+)- or a Mg(2+)-, dependent Ser/Thr phosphatase. Its activity is inhibited by EDTA and NaF, but not by okadaic acid, and is similar to that of PP2C phosphatase.
Topics: Amino Acid Sequence; Cloning, Molecular; Genes, Bacterial; Molecular Sequence Data; Multigene Family; Phosphoamino Acids; Phosphoprotein Phosphatases; Phosphorylation; Precipitin Tests; Protein Serine-Threonine Kinases; Pseudomonas aeruginosa; Recombinant Fusion Proteins; Reverse Transcriptase Polymerase Chain Reaction; Sequence Alignment; Serine; Threonine
PubMed: 10542161
DOI: 10.1128/JB.181.21.6615-6622.1999 -
Physiological Reviews Jan 2000This review deals with the influence of serine/threonine-specific protein phosphatases on the function of ion channels in the plasma membrane of excitable tissues.... (Review)
Review
This review deals with the influence of serine/threonine-specific protein phosphatases on the function of ion channels in the plasma membrane of excitable tissues. Particular focus is given to developments of the past decade. Most of the electrophysiological experiments have been performed with protein phosphatase inhibitors. Therefore, a synopsis is required incorporating issues from biochemistry, pharmacology, and electrophysiology. First, we summarize the structural and biochemical properties of protein phosphatase (types 1, 2A, 2B, 2C, and 3-7) catalytic subunits and their regulatory subunits. Then the available pharmacological tools (protein inhibitors, nonprotein inhibitors, and activators) are introduced. The use of these inhibitors is discussed based on their biochemical selectivity and a number of methodological caveats. The next section reviews the effects of these tools on various classes of ion channels (i.e., voltage-gated Ca(2+) and Na(+) channels, various K(+) channels, ligand-gated channels, and anion channels). We delineate in which cases a direct interaction between a protein phosphatase and a given channel has been proven and where a more complex regulation is likely involved. Finally, we present ideas for future research and possible pathophysiological implications.
Topics: Animals; Calcium Channels; Cell Membrane; Humans; Ion Channels; Phosphoprotein Phosphatases; Potassium Channels; Sodium Channels
PubMed: 10617768
DOI: 10.1152/physrev.2000.80.1.173 -
European Journal of Biochemistry Oct 1981Phosphoprotein phosphatase IA, which represents the major glycogen synthase phosphatase activity in rat liver cytosol, has been purified to apparent homogeneity by...
Phosphoprotein phosphatase IA, which represents the major glycogen synthase phosphatase activity in rat liver cytosol, has been purified to apparent homogeneity by chromatography on DEAE-cellulose, histone - Sepharose-4B and Sephadex G-100. The molecular weight of the purified enzyme was 40 000 by gel filtration and 48 000 by sodium dodecyl sulfate gel electrophoresis, Phosphatase IA is therefore a monomeric protein. When treated with 80% ethanol at room temperature, phosphatase IA underwent an inactivation which was totally prevented by 2 mM MgCl2. Catalytically, phosphatase IA has a preference for glycogen synthase D compared with phosphatases IB and II and obligatorily requires Mg2+ or Mn2+ for activity. Maximum activity was attained at 5 mM MgCl2. Since Mg2+ does not activate other phosphoprotein phosphatases in rat liver cytosol, we propose the term 'Mg2+-dependent glycogen synthase phosphatase' for phosphatase IA.
Topics: Animals; Chromatography; Cytosol; Enzyme Activation; Glycogen-Synthase-D Phosphatase; Liver; Magnesium; Male; Phosphoprotein Phosphatases; Rats; Rats, Inbred Strains
PubMed: 6273162
DOI: 10.1111/j.1432-1033.1981.tb05636.x -
Genes & Development May 2021The transcription cycle of RNA polymerase II (RNAPII) is governed at multiple points by opposing actions of cyclin-dependent kinases (CDKs) and protein phosphatases, in... (Review)
Review
The transcription cycle of RNA polymerase II (RNAPII) is governed at multiple points by opposing actions of cyclin-dependent kinases (CDKs) and protein phosphatases, in a process with similarities to the cell division cycle. While important roles of the kinases have been established, phosphatases have emerged more slowly as key players in transcription, and large gaps remain in understanding of their precise functions and targets. Much of the earlier work focused on the roles and regulation of sui generis and often atypical phosphatases-FCP1, Rtr1/RPAP2, and SSU72-with seemingly dedicated functions in RNAPII transcription. Decisive roles in the transcription cycle have now been uncovered for members of the major phosphoprotein phosphatase (PPP) family, including PP1, PP2A, and PP4-abundant enzymes with pleiotropic roles in cellular signaling pathways. These phosphatases appear to act principally at the transitions between transcription cycle phases, ensuring fine control of elongation and termination. Much is still unknown, however, about the division of labor among the PPP family members, and their possible regulation by or of the transcriptional kinases. CDKs active in transcription have recently drawn attention as potential therapeutic targets in cancer and other diseases, raising the prospect that the phosphatases might also present opportunities for new drug development. Here we review the current knowledge and outstanding questions about phosphatases in the context of the RNAPII transcription cycle.
Topics: Animals; Drug Delivery Systems; Humans; Phosphoprotein Phosphatases; RNA Polymerase II; Transcription, Genetic
PubMed: 33888562
DOI: 10.1101/gad.348315.121 -
The Journal of Biological Chemistry Aug 1989Neuromodulin (p57, GAP-43, F1, B-50) is a major neural-specific, calmodulin binding protein found in brain, spinal cord, and retina that is associated with membranes....
Neuromodulin (p57, GAP-43, F1, B-50) is a major neural-specific, calmodulin binding protein found in brain, spinal cord, and retina that is associated with membranes. Phosphorylation of neuromodulin by protein kinase C causes a significant reduction in its affinity for calmodulin (Alexander, K. A., Cimler, B. M., Meirer, K. E., and Storm, D. R. (1987) J. Biol. Chem. 262, 6108-6113). It has been proposed that neuromodulin may function to bind and concentrate calmodulin at specific sites within neurons and that activation of protein kinase C causes the release of free calmodulin at high concentrations near its target proteins. It was the goal of this study to determine whether bovine brain contains a phosphoprotein phosphatase that will utilize phosphoneuromodulin as a substrate. Phosphatase activity for phosphoneuromodulin was partially purified from a bovine brain extract using DEAE-Sephacel and Sephacryl S-200 gel filtration chromatography. The neuromodulin phosphatase activity was resolved into two peaks by Affi-Gel Blue chromatography. One of these phosphatases, which represented approximately 60% of the total neuromodulin phosphatase activity, was tentatively identified as calcineurin by its requirement for Ca2+ and calmodulin (CaM) and inhibition of its activity by chlorpromazine. Therefore, bovine brain calcineurin was purified to homogeneity and examined for its phosphatase activity against bovine phosphoneuromodulin. Calcineurin rapidly dephosphorylated phosphoneuromodulin in the presence of micromolar Ca2+ and 3 microM CaM. The apparent Km and Vmax for the dephosphorylation of neuromodulin, measured in the presence of micromolar Ca2+ and 2 microM CaM, were 2.5 microM and 70 nmol Pi/mg/min, respectively, compared to a Km and Vmax of 4 microM and 55 nmol Pi/mg/min, respectively, for myosin light chain under the same conditions. Dephosphorylation of neuromodulin by calcineurin was stimulated 50-fold by calmodulin in the presence of micromolar free Ca2+. Half-maximal stimulation was observed at a calmodulin concentration of 0.5 microM. We propose that phosphoneuromodulin may be a physiologically important substrate for calcineurin and that calcineurin and protein kinase C may regulate the levels of free calmodulin available in neurons.
Topics: Animals; Brain; Calcineurin; Calmodulin-Binding Proteins; Catalysis; Cattle; GAP-43 Protein; Nerve Tissue Proteins; Phosphoprotein Phosphatases; Phosphorylation; Substrate Specificity
PubMed: 2546935
DOI: No ID Found -
European Journal of Biochemistry Mar 1981The MgATP-dependent phosphorylase phosphatase was found to have a broad substrate specificity. Its activity against all phosphoproteins tested was dependent upon... (Comparative Study)
Comparative Study
The MgATP-dependent phosphorylase phosphatase was found to have a broad substrate specificity. Its activity against all phosphoproteins tested was dependent upon preincubation with the activating factor FA and MgATP. The enzyme dephosphorylated and inactivated phosphorylase kinase and inhibitor 1, and dephosphorylated and activated glycogen synthase and acetyl-CoA carboxylase. Glycogen synthase was dephosphorylated at similar rates whether it had been phosphorylated by cyclic-AMP-dependent protein kinase, phosphorylase kinase or glycogen synthase kinase 3. The enzyme also catalysed the dephosphorylation of ATP citrate lyase, initiation factor eIF-2, and troponin I. The properties of the MgATP-dependent protein phosphatase from either dog liver or rabbit skeletal muscle showed a remarkable similarity to highly purified preparations of protein phosphatase 1 from rabbit skeletal muscle. The relative activities of the two enzymes against all phosphoproteins tested was very similar. Both enzymes dephosphorylated the beta-subunit of phosphorylase kinase 40-fold faster than the alpha-subunit, and both enzymes were inhibited by identical concentrations of the two proteins termed inhibitor 1 and inhibitor 2, which inhibit protein phosphatase 1 specifically. These results demonstrate that the MgATP-dependent protein phosphatase is a type-1 protein phosphatase, and is distinct from type-2 protein phosphatases which dephosphorylate the alpha-subunit of phosphorylase kinase and are unaffected by inhibitor 1 and inhibitor 2. The possibility that the MgATP-dependent protein phosphatase is an inactive form of protein phosphatase 1 and that both proteins share the same catalytic subunit is discussed.
Topics: Adenosine Triphosphate; Animals; Catalysis; Dogs; Enzyme Activation; Liver; Muscles; Phosphoprotein Phosphatases; Protein Phosphatase 1; Rabbits; Substrate Specificity
PubMed: 6262081
DOI: 10.1111/j.1432-1033.1981.tb06217.x -
The FEBS Journal Jan 2013Now, as we try to define how phosphorylation-dependent cell signalling pathways are integrated at the level of the whole organism, it is important to understand fully...
Now, as we try to define how phosphorylation-dependent cell signalling pathways are integrated at the level of the whole organism, it is important to understand fully the function of the protein phosphatases. This Special Issue of FEBS Journal presents articles that reflect the theme of specificity in protein phosphatase function and emphasize the critical role played by these enzymes in the regulation of signal transduction.
Topics: Animals; Binding Sites; Humans; Isoenzymes; Models, Molecular; Phosphoprotein Phosphatases; Phosphorylation; Protein Binding; Protein Structure, Tertiary; Protein Subunits; Signal Transduction
PubMed: 23279779
DOI: 10.1111/febs.12098 -
FEBS Letters Sep 1981
Topics: Animals; Drug Stability; Enzyme Reactivators; Hot Temperature; Liver; Magnesium; Magnesium Chloride; Phosphoprotein Phosphatases; Phosphorylase Phosphatase; Pyruvate Kinase; Rats; Trypsin
PubMed: 6271587
DOI: 10.1016/0014-5793(81)81156-8 -
The Journal of Biological Chemistry Nov 1985A high molecular weight phosphoprotein phosphatase was purified from rabbit liver using high speed centrifugation, acid precipitation, ammonium sulfate fractionation,...
A high molecular weight phosphoprotein phosphatase was purified from rabbit liver using high speed centrifugation, acid precipitation, ammonium sulfate fractionation, chromatography on DEAE-cellulose, Sepharose-histone, and Bio-Gel A-0.5m. The purified enzyme showed a single band on a nondenaturing polyacrylamide anionic disc gel which was associated with the enzyme activity. The enzyme was made up of equimolar concentrations of two subunits whose molecular weights were 58,000 (range 58,000-62,000) and 35,000 (range 35,000-38,000). Two other polypeptides (Mr 76,000 and 27,000) were also closely associated with our enzyme preparation, but their roles, if any, in phosphatase activity are not known. The optimum pH for the reaction was 7.5-8.0. Km value of phosphoprotein phosphatase for phosphorylase a was 0.10-0.12 mg/ml. Freezing and thawing of the enzyme in the presence of 0.2 M beta-mercaptoethanol caused an activation (100-140%) of phosphatase activity with a concomitant partial dissociation of the enzyme into a Mr 35,000 catalytic subunit. Divalent cations (Mg2+, Mn2+, and Co2+) and EDTA were inhibitory at concentrations higher than 1 mM. Spermine and spermidine were also found to be inhibitory at 1 mM concentrations. The enzyme was inhibited by nucleotides (ATP, ADP, AMP), PPi, Pi, and NaF; the degree of inhibition was different with each compound and was dependent on their concentrations employed in the assay. Among various types of histones examined, maximum activation of phosphoprotein phosphatase activity was observed with type III and type V histone (Sigma). Further studies with type III histone indicated that it increased both the Km for phosphorylase a and the Vmax of the dephosphorylation reaction. Purified liver phosphatase, in addition to the dephosphorylation of phosphorylase a, also catalyzed the dephosphorylation of 32P-labeled phosphorylase kinase, myosin light chain, myosin, histone III-S, and myelin basic protein. The effects of Mn2+, KCl, and histone III-S on phosphatase activity were variable depending on the substrate used.
Topics: Animals; Caffeine; Chromatography; Dithioerythritol; Drug Stability; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Freezing; Histones; Hydrogen-Ion Concentration; Kinetics; Liver; Mercaptoethanol; Molecular Weight; Phosphoprotein Phosphatases; Phosphorylase a; Rabbits; Substrate Specificity
PubMed: 2997204
DOI: No ID Found -
Microbiology (Reading, England) Aug 2005Phosphoprotein phosphatases encoded by the prpA and prpB genes function in signal transduction pathways for degradation of misfolded proteins in the extracytoplasmic...
Phosphoprotein phosphatases encoded by the prpA and prpB genes function in signal transduction pathways for degradation of misfolded proteins in the extracytoplasmic compartments of Escherichia coli. In order to trace the evolution of prp genes and assess their roles in other enteric pathogens, the structure and distribution of these genes among closely related Shigella subgroups were studied. PCR amplification, probe hybridization studies and DNA sequencing were used to determine the prp genotypes of 58 strains from the four Shigella subgroups, Dysenteriae, Boydii, Sonnei and Flexneri. It was found that the prp alleles among Shigella subgroups were extremely susceptible to gene inactivation and that the mutations involved in prp allele inactivation were varied. They included IS insertions, gene replacement by an IS element, a small deletion within the gene or large deletion engulfing the entire gene region, and base substitutions that generated premature termination codons. As a result, of 58 strains studied, only eight (14 %) possessed intact prpA and prpB genes. Of the Shigella strains examined, 76 % (44/58) showed at least one of the prp alleles inactivated by one or more IS elements, including IS1, IS4, IS600 and IS629. Phylogenetic analysis revealed that IS elements have been independently acquired in multiple lineages of Shigella, suggesting that loss of functional alleles has been advantageous during Shigella strain evolution.
Topics: DNA Transposable Elements; Molecular Sequence Data; Phosphoprotein Phosphatases; Phylogeny; Shigella; Signal Transduction
PubMed: 16079345
DOI: 10.1099/mic.0.27990-0