-
Chemistry & Biology Jan 2000Reversible protein phosphorylation is a major regulatory mechanism of intracellular signal transduction. Protein phosphatase 1 (PP1) is one of four major types of... (Review)
Review
Reversible protein phosphorylation is a major regulatory mechanism of intracellular signal transduction. Protein phosphatase 1 (PP1) is one of four major types of serine-threonine phosphatases mediating signaling pathways, but the means by which its activity is modulated has only recently begun to come into focus.
Topics: Amino Acid Sequence; Animals; Gene Expression Regulation, Enzymologic; Humans; Molecular Sequence Data; Phosphoprotein Phosphatases; Protein Phosphatase 1; Regulatory Sequences, Nucleic Acid
PubMed: 10662690
DOI: 10.1016/s1074-5521(00)00069-7 -
The Journal of Biological Chemistry Nov 1995Nucleoside-diphosphate kinase (ATP:nucleoside-diphosphate phosphotransferase, EC 2.7.4.6; NDP kinase) is an important enzyme for the maintenance of the correct cellular... (Comparative Study)
Comparative Study
Nucleoside-diphosphate kinase (ATP:nucleoside-diphosphate phosphotransferase, EC 2.7.4.6; NDP kinase) is an important enzyme for the maintenance of the correct cellular levels of nucleoside triphosphates (NTPs) and their deoxy derivatives (dNTPs) and is involved in the regulation of cellular development. The enzyme is under the dual control of algR2 and algH in Pseudomonas aeruginosa. We report here the purification and characterization of a protein that dephosphorylates the phosphorylated intermediate form of the P. aeruginosa NDP kinase (Ndk). Dephosphorylation of Ndk phosphate leads to loss of its enzymatic activity. The 10.1-kDa polypeptide shows 77% homology at the N terminus with the Spo0E phosphatase, identified as a negative regulator of sporulation in Bacillus subtilis and 66% with the human Bax protein, identified as an effector of programmed cell death. The phosphatase termed Npp showed varied specificity toward phosphorylated Ndks from different sources including human erythrocyte Ndk phosphate. Its activity toward other histidine phosphates such as CheA or the alpha-subunit of succinyl-CoA synthetase or phosphoesters such as p-nitrophenyl phosphate was quite limited. Npp was stable at room temperature up to 2 h and required Mg2+ for activity. The presence of a phosphatase capable of dephosphorylating the phosphorylated form of P. aeruginosa Ndk represents an interesting and efficient mode of post-translational modification of an enzyme crucial to cellular development.
Topics: Adenosine Triphosphate; Amino Acid Sequence; Bacterial Proteins; Chromatography, Gel; Chromatography, Ion Exchange; Enzyme Stability; Humans; Kinetics; Molecular Sequence Data; Molecular Weight; Nucleoside-Diphosphate Kinase; Peptide Fragments; Phosphoprotein Phosphatases; Phosphorus Radioisotopes; Phosphorylation; Pseudomonas aeruginosa; Sequence Homology, Amino Acid; Sigma Factor; Substrate Specificity; Transcription Factors
PubMed: 7499321
DOI: 10.1074/jbc.270.47.28246 -
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 -
Molecular Plant-microbe Interactions :... Aug 2007Sclerotinia sclerotiorum is a necrotrophic, omnivorous plant pathogen with worldwide distribution. Sclerotia of S. sclerotiorum are pigmented, multihyphal structures...
Sclerotinia sclerotiorum is a necrotrophic, omnivorous plant pathogen with worldwide distribution. Sclerotia of S. sclerotiorum are pigmented, multihyphal structures that play a central role in the life and infection cycles of this pathogen. Plant infection depends on the formation of melanin-rich infection cushions, and secretion of hydrolytic enzymes and oxalic acid. Type 2A Ser/Thr phosphatases (PP2As) are involved in the regulation of a variety of cellular process. In the presence of cantharidin, a PP2A-specific inhibitor, hyphal elongation and sclerotia numbers were impaired whereas sclerotial size increased. We partially inactivated PP2A by antisense expression of the gene (pph1) encoding the PP2A catalytic subunit. When antisense expression was induced, almost complete cessation of fungal growth was observed, indicative of a crucial role for PP2A in fungal growth. RNAi-based gene silencing was employed to alter the expression of the 55-kDa R2 (B regulatory subunit). Isolates in which rgb1 RNA levels were decreased were slow growing, but viable. Melanin biosynthesis, infection-cushion production, and pathogenesis were significantly impaired in the rgb1 mutants, yet theses mutants were pathogenic on wounded leaves. Reduced ERK (extracellular signal-regulated kinases)-like mitogen-activated protein kinase (MAPK) function conferred a reduction in NADPH oxidase and PP2A activity levels, suggesting a functional link between MAPK, reactive oxygen species, and PP2A activity in S. sclerotiorum.
Topics: Ascomycota; Cantharidin; Enzyme Inhibitors; Fungal Proteins; Hyphae; MAP Kinase Signaling System; NADPH Oxidases; Phosphoprotein Phosphatases; RNA Interference; Reproduction, Asexual; Virulence
PubMed: 17722698
DOI: 10.1094/MPMI-20-8-0944 -
Frontiers in Bioscience : a Journal and... Mar 1999Protein phosphatase-1, originally studied as phosphorylase phosphatase, is one of the major ser/thr protein phosphatases. It has a long history and a complex enzymology.... (Review)
Review
Protein phosphatase-1, originally studied as phosphorylase phosphatase, is one of the major ser/thr protein phosphatases. It has a long history and a complex enzymology. It consists of a catalytic subunit of 37 kDa, which is bound to a number of different regulatory or targeting subunits. These are believed to restrict its activity to its immediate microenvironment and thus define its specificity, as well as acting to regulate phosphatase activity. The existence of multiple protein phosphatase-1 binding proteins provides the mechanism whereby phosphatase-1 activity can be involved in a diverse range of cellular functions, and reflects a novel strategy for its evolutionary development.
Topics: Amino Acid Sequence; Animals; Calcineurin; Carrier Proteins; Catalytic Domain; Cloning, Molecular; Enzyme Activation; Glycogen; Holoenzymes; Humans; Metalloproteins; Models, Molecular; Molecular Sequence Data; Mutagenesis; Phosphoprotein Phosphatases; Protein Phosphatase 1; Recombinant Proteins; Structure-Activity Relationship
PubMed: 10077543
DOI: 10.2741/lee -
Journal of Visualized Experiments : JoVE Apr 2022Most cellular processes are regulated by dynamic protein phosphorylation. More than three-quarters of proteins are phosphorylated, and phosphoprotein phosphatases (PPPs)...
Most cellular processes are regulated by dynamic protein phosphorylation. More than three-quarters of proteins are phosphorylated, and phosphoprotein phosphatases (PPPs) coordinate over 90% of all cellular serine/threonine dephosphorylation. Deregulation of protein phosphorylation has been implicated in the pathophysiology of various diseases, including cancer and neurodegeneration. Despite their widespread activity, the molecular mechanisms controlling PPPs and those controlled by PPPs are poorly characterized. Here, a proteomic approach termed phosphatase inhibitor beads and mass spectrometry (PIB-MS) is described to identify and quantify PPPs, their posttranslational modifications, and their interactors in as little as 12 h using any cell line or tissue. PIB-MS utilizes a non-selective PPP inhibitor, microcystin-LR (MCLR), immobilized on sepharose beads to capture and enrich endogenous PPPs and their associated proteins (termed the PPPome). This method does not require the exogenous expression of tagged versions of PPPs or the use of specific antibodies. PIB-MS offers an innovative way to study the evolutionarily conserved PPPs and expand our current understanding of dephosphorylation signaling.
Topics: Mass Spectrometry; Phosphoprotein Phosphatases; Phosphorylation; Protein Processing, Post-Translational; Proteomics
PubMed: 35575520
DOI: 10.3791/63805 -
Cell Systems Apr 2017Coordinated activities of protein kinases and phosphatases ensure phosphorylation homeostasis, which, when perturbed, can instigate diseases, including cancer. Yet, in...
Coordinated activities of protein kinases and phosphatases ensure phosphorylation homeostasis, which, when perturbed, can instigate diseases, including cancer. Yet, in contrast to kinases, much less is known about protein phosphatase functions and their interactions and complexes. Here, we used quantitative affinity proteomics to assay protein-protein interactions for 54 phosphatases distributed across the three major protein phosphatase families, with additional analysis of their 12 co-factors. We identified 838 high-confidence interactions, of which 631, to our knowledge, have not been reported before. We show that inhibiting the activity of phosphatases PP1 and PP2A by okadaic acid disrupts their specific interactions, supporting the potential of therapeutics that target these proteins. Additional analyses revealed candidate physical and functional interaction links to phosphatase-based regulation of several signaling pathways and to human cancer. Our study provides an initial glimpse of the protein interaction landscape of phosphatases and their functions in cellular regulation.
Topics: Humans; Mass Spectrometry; Neoplasms; Phosphoprotein Phosphatases; Phosphorylation; Protein Interaction Mapping; Protein Interaction Maps; Proteome; Proteomics; Signal Transduction; Tandem Affinity Purification
PubMed: 28330616
DOI: 10.1016/j.cels.2017.02.011 -
Biochemical Pharmacology Oct 2000Type 2A protein phosphatase (PP2A) comprises a diverse family of phosphoserine- and phosphothreonine-specific enzymes ubiquitously expressed in eukaryotic cells. Common... (Review)
Review
Type 2A protein phosphatase (PP2A) comprises a diverse family of phosphoserine- and phosphothreonine-specific enzymes ubiquitously expressed in eukaryotic cells. Common to all forms of PP2A is a catalytic subunit (PP2Ac) which can form two distinct complexes, one with a structural subunit termed PR65/A and another with an alpha4 protein. The PR65/A-PP2Ac dimer may further associate with a regulatory subunit and form a trimeric holoenzyme. To date, three distinct families of regulatory subunits, which control substrate selectivity and phosphatase activity and target PP2A holoenzymes to their substrates, have been identified. Other molecular mechanisms that regulate PP2Ac function include phosphorylation, carboxyl methylation, inhibition by intracellular protein inhibitors (I(1)(PP2A) and I(2)(PP2A)), and stimulation by ceramide. PP2A dephosphorylates many proteins in vitro, but in vivo protein kinases and transcription factors appear to represent two major sets of substrates. Several natural compounds can inhibit PP2A activity and are used to study its function. Mutations in genes encoding PR65/A subunits have been identified in several different human cancers and the PP2A inhibitor, termed fostriecin, is being tested as an anticancer drug. Thus, a more thorough understanding of PP2A structure and function may lead to the development of novel strategies against human diseases.
Topics: Cell Cycle; Enzyme Activation; Phosphoprotein Phosphatases; Protein Conformation; Protein Kinases; Signal Transduction; Substrate Specificity; Toxins, Biological; Transcription Factors
PubMed: 11007961
DOI: 10.1016/s0006-2952(00)00424-x -
Journal of Dairy Science Feb 1988An acid phosphatase has been partially purified from lactating bovine mammary gland. Properties of this enzyme were compared with those of a well-characterized...
An acid phosphatase has been partially purified from lactating bovine mammary gland. Properties of this enzyme were compared with those of a well-characterized phosphoprotein phosphatase from bovine spleen. The two enzymes were similar in their activation by sulfhydryl reagents and inhibition by metal chelating agents. Both enzymes rapidly hydrolyze ATP and aromatic phosphates and are relatively inactive toward alkyl phosphates; both are tartrate-resistant phosphatases. The mammary enzyme has a low Michaelis constant for alpha s1-casein (42 microM), and thus, like the spleen enzyme, appears to be a phosphoprotein phosphatase. Finally, the spleen and mammary enzymes displayed reactivity toward phosphotyrosine, a model substrate for phosphotyrosyl protein phosphatase. Thus, the phosphatases from spleen and mammary gland are quite similar in reactivity and could possibly be similar in function.
Topics: Acid Phosphatase; Animals; Cattle; Female; Lactation; Mammary Glands, Animal; Phosphoprotein Phosphatases; Phosphotyrosine; Pregnancy; Tyrosine
PubMed: 2454249
DOI: 10.3168/jds.S0022-0302(88)79560-0 -
The Biochemical Journal Feb 2001Protein phosphatase 2A (PP2A) comprises a family of serine/threonine phosphatases, minimally containing a well conserved catalytic subunit, the activity of which is... (Review)
Review
Protein phosphatase 2A (PP2A) comprises a family of serine/threonine phosphatases, minimally containing a well conserved catalytic subunit, the activity of which is highly regulated. Regulation is accomplished mainly by members of a family of regulatory subunits, which determine the substrate specificity, (sub)cellular localization and catalytic activity of the PP2A holoenzymes. Moreover, the catalytic subunit is subject to two types of post-translational modification, phosphorylation and methylation, which are also thought to be important regulatory devices. The regulatory ability of PTPA (PTPase activator), originally identified as a protein stimulating the phosphotyrosine phosphatase activity of PP2A, will also be discussed, alongside the other regulatory inputs. The use of specific PP2A inhibitors and molecular genetics in yeast, Drosophila and mice has revealed roles for PP2A in cell cycle regulation, cell morphology and development. PP2A also plays a prominent role in the regulation of specific signal transduction cascades, as witnessed by its presence in a number of macromolecular signalling modules, where it is often found in association with other phosphatases and kinases. Additionally, PP2A interacts with a substantial number of other cellular and viral proteins, which are PP2A substrates, target PP2A to different subcellular compartments or affect enzyme activity. Finally, the de-regulation of PP2A in some specific pathologies will be touched upon.
Topics: Animals; Cell Division; Phosphoprotein Phosphatases; Protein Phosphatase 2; Signal Transduction
PubMed: 11171037
DOI: 10.1042/0264-6021:3530417