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MBio May 2021The circadian clock controls the phosphorylation and activity of eukaryotic translation initiation factor 2α (eIF2α). In , the clock drives a daytime peak in the...
The circadian clock controls the phosphorylation and activity of eukaryotic translation initiation factor 2α (eIF2α). In , the clock drives a daytime peak in the activity of the eIF2α kinase CPC-3, the homolog of yeast and mammalian GCN2 kinase. This leads to increased levels of phosphorylated eIF2α (P-eIF2α) and reduced mRNA translation initiation during the day. We hypothesized that rhythmic eIF2α activity also requires dephosphorylation of P-eIF2α at night by phosphatases. In support of this hypothesis, we show that mutation of PPP-1, a homolog of the yeast eIF2α phosphatase GLC7, leads to high and arrhythmic P-eIF2α levels, while maintaining core circadian oscillator function. PPP-1 levels are clock-controlled, peaking in the early evening, and rhythmic PPP-1 levels are necessary for rhythmic P-eIF2α accumulation. Deletion of the N terminus of eIF2γ, the region necessary for eIF2γ interaction with GLC7 in yeast, led to high and arrhythmic P-eIF2α levels. These data supported that eIF2γ functions to recruit PPP-1 to dephosphorylate eIF2α at night. Thus, in addition to the activity of CPC-3 kinase, circadian clock regulation of eIF2α activity requires dephosphorylation by PPP-1 phosphatase at night. These data show how the circadian clock controls the activity a central regulator of translation, critical for cellular metabolism and growth control, through the temporal coordination of phosphorylation and dephosphorylation events. Circadian clock control of mRNA translation contributes to the daily cycling of a significant proportion of the cellular protein synthesis, but how this is accomplished is not understood. We discovered that the clock in the model fungus regulates rhythms in protein synthesis by controlling the phosphorylation and dephosphorylation of a conserved translation initiation factor eIF2α. During the day, eIF2α is phosphorylated and inactivated by CPC-3 kinase. At night, a clock-controlled phosphatase, PPP-1, dephosphorylates and activates eIF2α, leading to increased nighttime protein synthesis. Translation requires significant cellular energy; thus, partitioning translation to the night by the clock provides a mechanism to coordinate energy metabolism with protein synthesis and cellular growth.
Topics: Circadian Clocks; Eukaryotic Initiation Factor-2; Fungal Proteins; Neurospora crassa; Phosphorylation; Protein Biosynthesis; Protein Phosphatase 1
PubMed: 34006661
DOI: 10.1128/mBio.00871-21 -
Molecular Biology of the Cell Nov 2007Fission yeast mitogen-activated protein kinase (MAPK) Pmk1p is involved in morphogenesis, cytokinesis, and ion homeostasis as part of the cell integrity pathway, and it...
Fission yeast mitogen-activated protein kinase (MAPK) Pmk1p is involved in morphogenesis, cytokinesis, and ion homeostasis as part of the cell integrity pathway, and it becomes activated under multiple stresses, including hyper- or hypotonic conditions, glucose deprivation, cell wall-damaging compounds, and oxidative stress. The only protein phosphatase known to dephosphorylate and inactivate Pmk1p is Pmp1p. We show here that the stress-activated protein kinase (SAPK) pathway and its main effector, Sty1p MAPK, are essential for proper deactivation of Pmk1p under hypertonic stress in a process regulated by Atf1p transcription factor. We demonstrate that tyrosine phosphatases Pyp1p and Pyp2p, and serine/threonine phosphatase Ptc1p, that negatively regulate Sty1p activity and whose expression is dependent on Sty1p-Atf1p function, are involved in Pmk1p dephosphorylation under osmostress. Pyp1p and Ptc1p, in addition to Pmp1p, also control the basal level of MAPK Pmk1p activity in growing cells and associate with, and dephosphorylate Pmk1p both in vitro and in vivo. Our results with Ptc1p provide the first biochemical evidence for a PP2C-type phosphatase acting on more than one MAPK in yeast cells. Importantly, the SAPK-dependent down-regulation of Pmk1p through Pyp1p, Pyp2p, and Ptc1p was not complete, and Pyp1p and Ptc1p phosphatases are able to negatively regulate MAPK Pmk1p activity by an alternative regulatory mechanism. Our data also indicate that Pmk1p phosphorylation oscillates as a function of the cell cycle, peaking at cell separation during cytokinesis, and that Pmp1p phosphatase plays a main role in regulating this process.
Topics: Cell Cycle; Down-Regulation; Enzyme Activation; Gene Deletion; Gene Expression Regulation, Fungal; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Osmotic Pressure; Phosphoprotein Phosphatases; Phosphorylation; Phosphothreonine; Protein Binding; Schizosaccharomyces; Schizosaccharomyces pombe Proteins
PubMed: 17761528
DOI: 10.1091/mbc.e07-05-0484 -
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 Journal of Neuroscience : the... Sep 1997Taurine is known to be involved in many important physiological functions. Here we report that both in vivo and in vitro the taurine-synthesizing enzyme in the brain,...
Taurine is known to be involved in many important physiological functions. Here we report that both in vivo and in vitro the taurine-synthesizing enzyme in the brain, namely cysteine sulfinic acid decarboxylase (CSAD), is activated when phosphorylated and inhibited when dephosphorylated. Furthermore, protein kinase C and protein phosphatase 2C have been identified as the enzymes responsible for phosphorylation and dephosphorylation of CSAD, respectively. In addition, the effect of neuronal depolarization on CSAD activity and 32P incorporation into CSAD in neuronal cultures is also included. A model to link neuronal excitation and CSAD activation by a Ca2+-dependent protein kinase is proposed.
Topics: Animals; Carboxy-Lyases; Cells, Cultured; Enzyme Activation; Glutamate Decarboxylase; Models, Chemical; Phosphoprotein Phosphatases; Phosphorylation; Protein Kinase C; Protein Kinases; Protein Phosphatase 2; Protein Phosphatase 2C; Proteins; Saccharomyces cerevisiae Proteins; Swine; Synaptosomes; Taurine
PubMed: 9278530
DOI: 10.1523/JNEUROSCI.17-18-06947.1997 -
BioRxiv : the Preprint Server For... May 2024Calcineurin (CN), the only Ca -calmodulin activated protein phosphatase, dephosphorylates substrates within membrane-associated Ca microdomains. CN binds to substrates...
Calcineurin (CN), the only Ca -calmodulin activated protein phosphatase, dephosphorylates substrates within membrane-associated Ca microdomains. CN binds to substrates and regulators via short linear motifs (SLIMs), PxIxIT and LxVP. PxIxIT binding to CN is Ca independent and affects its distribution, while LxVP associates only with the active enzyme and promotes catalysis. 31 human proteins contain one or more composite 'LxVPxIxIT' motifs, whose functional properties have not been examined. Here we report studies of calcimembrin/C16orf74 (CLMB), a largely uncharacterized protein containing a composite motif that binds and directs CN to membranes. We demonstrate that CLMB associates with membranes via N-myristoylation and dynamic S-acylation and is dephosphorylated by CN on Thr44. The LxVP and PxIxIT portions of the CLMB composite sequence, together with Thr44 phosphorylation, confer high affinity PxIxIT-mediated binding to CN (KD∼8.9 nM) via an extended, LxVPxIxITxx(p)T sequence. This binding promotes CLMB-based targeting of CN to membranes, but also protects Thr44 from dephosphorylation. Thus, we propose that CN dephosphorylates CLMB in multimeric complexes, where one CLMB molecule recruits CN to membranes via PxIxIT binding, allowing others to engage through their LxVP motif for dephosphorylation. This unique mechanism makes dephosphorylation sensitive to CLMB:CN ratios and is supported by and analyses. CLMB overexpression is associated with poor prognoses for several cancers, suggesting that it promotes oncogenesis by shaping CN signaling.
PubMed: 38798520
DOI: 10.1101/2024.05.12.593783 -
The Journal of Biological Chemistry Apr 2023To cope with an increased external osmolarity, the budding yeast Saccharomyces cerevisiae activates the Hog1 mitogen-activated protein kinase (MAPK) through the...
To cope with an increased external osmolarity, the budding yeast Saccharomyces cerevisiae activates the Hog1 mitogen-activated protein kinase (MAPK) through the high-osmolarity glycerol (HOG) pathway, which governs adaptive responses to osmostress. In the HOG pathway, two apparently redundant upstream branches, termed SLN1 and SHO1, activate cognate MAP3Ks (MAPKK kinase) Ssk2/22 and Ste11, respectively. These MAP3Ks, when activated, phosphorylate and thus activate the Pbs2 MAP2K (MAPK kinase), which in turn phosphorylates and activates Hog1. Previous studies have shown that protein tyrosine phosphatases and the serine/threonine protein phosphatases type 2C negatively regulate the HOG pathway to prevent its excessive and inappropriate activation, which is detrimental to cell growth. The tyrosine phosphatases Ptp2 and Ptp3 dephosphorylate Hog1 at Tyr-176, whereas the protein phosphatase type 2Cs Ptc1 and Ptc2 dephosphorylate Hog1 at Thr-174. In contrast, the identities of phosphatases that dephosphorylate Pbs2 remained less clear. Here, we examined the phosphorylation status of Pbs2 at the activating phosphorylation sites Ser-514 and Thr-518 (S514 and T518) in various mutants, both in the unstimulated and osmostressed conditions. Thus, we found that Ptc1-Ptc4 collectively regulate Pbs2 negatively, but each Ptc acts differently to the two phosphorylation sites in Pbs2. T518 is predominantly dephosphorylated by Ptc1, while S514 can be dephosphorylated by any of Ptc1-4 to an appreciable extent. We also show that Pbs2 dephosphorylation by Ptc1 requires the adaptor protein Nbp2 that recruits Ptc1 to Pbs2, thus highlighting the complex processes involved in regulating adaptive responses to osmostress.
Topics: Glycerol; Intracellular Signaling Peptides and Proteins; MAP Kinase Kinase Kinases; Mitogen-Activated Protein Kinase Kinases; Osmolar Concentration; Phosphoprotein Phosphatases; Phosphorylation; Protein Kinases; Protein Phosphatase 2C; Protein Tyrosine Phosphatases; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Signal Transduction
PubMed: 36870684
DOI: 10.1016/j.jbc.2023.104569 -
Journal of Biochemistry Dec 1995We have reported that many sites of tau in fetal brain (fetal-tau) as well as in paired helical filaments (PHF-tau) are phosphorylated. In the present study, we used...
We have reported that many sites of tau in fetal brain (fetal-tau) as well as in paired helical filaments (PHF-tau) are phosphorylated. In the present study, we used site-specific antibodies and peptide mapping to examine protein phosphatases involved in dephosphorylation of fetal-tau and PHF-tau. Immunoblot analysis and electrophoretic mobility showed that protein phosphatases 1 and 2A and calcineurin could dephosphorylate fetal-tau and PHF-tau. Phosphoserines 199, 202, 396, and 413 and phosphothreonine 231, numbered according to the longest human tau isoform, were dephosphorylated, as shown by the immunoblot analysis. Phosphoserine 422 was dephosphorylated by protein phosphatase 2A and calcineurin, but not by protein phosphatase 1. Peptide mapping with Achromobacter lyticus protease 1 showed that phosphoserines 199, 202, 235, and 396 and phosphothreonine 231 were dephosphorylated by protein phosphatases. Fetal-tau was more rapidly dephosphorylated by protein phosphatase 2A and calcineurin than PHF-tau. Interestingly, PHF-tau which had not been solubilized with guanidine HCl was little dephosphorylated by protein phosphatases. Thus, PHF-tau in neurofibrillary tangles of Alzheimer's disease brain is likely to be resistant to dephosphorylation by protein phosphatases.
Topics: Amino Acid Sequence; Animals; Animals, Newborn; Brain; Calcineurin; Calmodulin; Calmodulin-Binding Proteins; Cattle; Electrophoresis, Polyacrylamide Gel; Fetus; Immunoblotting; Kinetics; Molecular Sequence Data; Peptide Fragments; Peptide Mapping; Phosphopeptides; Phosphoprotein Phosphatases; Protein Phosphatase 1; Protein Phosphatase 2; Protein Structure, Secondary; Rats; Recombinant Proteins; Solubility; tau Proteins
PubMed: 8720139
DOI: 10.1093/oxfordjournals.jbchem.a125011 -
FEMS Microbiology Reviews Aug 2002Major advances have recently occurred in our understanding of GATA factor-mediated, nitrogen catabolite repression (NCR)-sensitive gene expression in Saccharomyces... (Review)
Review
Major advances have recently occurred in our understanding of GATA factor-mediated, nitrogen catabolite repression (NCR)-sensitive gene expression in Saccharomyces cerevisiae. Under nitrogen-rich conditions, the GATA family transcriptional activators, Gln3 and Gat1, form complexes with Ure2, and are localized to the cytoplasm, which decreases NCR-sensitive expression. Under nitrogen-limiting conditions, Gln3 and Gat1 are dephosphorylated, move from the cytoplasm to the nucleus, in wild-type but not rna1 and srp1 mutants, and increase expression of NCR-sensitive genes. 'Induction' of NCR-sensitive gene expression and dephosphorylation of Gln3 (and Ure2 in some laboratories) when cells are treated with rapamycin implicates the Tor1/2 signal transduction pathway in this regulation. Mks1 is posited to be a negative regulator of Ure2, positive regulator of retrograde gene expression and to be itself negatively regulated by Tap42. In addition to Tap42, phosphatases Sit4 and Pph3 are also argued by some to participate in the regulatory pathway. Although a treasure trove of information has recently become available, much remains unknown (and sometimes controversial) with respect to the precise biochemical functions and regulatory pathway connections of Tap42, Sit4, Pph3, Mks1 and Ure2, and how precisely Gln3 and Gat1 are prevented from entering the nucleus. The purpose of this review is to provide background information needed by students and investigators outside of the field to follow and evaluate the rapidly evolving literature in this exciting field.
Topics: Antifungal Agents; DNA-Binding Proteins; Fungal Proteins; Gene Expression Regulation, Fungal; Glutathione Peroxidase; Nitrogen; Oligopeptides; Prions; Pyrrolidonecarboxylic Acid; Repressor Proteins; Ribosomal Protein S6 Kinases, 90-kDa; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Signal Transduction; Sirolimus; Transcription Factors
PubMed: 12165425
DOI: 10.1111/j.1574-6976.2002.tb00612.x -
Molecular and Cellular Biology May 1993Cyclic AMP (cAMP)-dependent protein kinase A (PKA) stimulates the transcription of many eucaryotic genes by catalyzing the phosphorylation of the cAMP-regulatory element...
Cyclic AMP (cAMP)-dependent protein kinase A (PKA) stimulates the transcription of many eucaryotic genes by catalyzing the phosphorylation of the cAMP-regulatory element binding protein (CREB). Conversely, the attenuation or inhibition of cAMP-stimulated gene transcription would require the dephosphorylation of CREB by a nuclear protein phosphatase. In HepG2 cells treated with the protein serine/threonine (Ser/Thr) phosphatase inhibitor okadaic acid, dibutyryl-cAMP-stimulated transcription from the phosphoenolpyruvate carboxykinase (PEPCK) promoter was enhanced over the level of PEPCK gene transcription observed in cells treated with dibutyryl-cAMP alone. This process was mediated, at least in part, by a region of the PEPCK promoter that binds CREB. Likewise, okadaic acid prevents the dephosphorylation of PKA-phosphorylated CREB in rat liver nuclear extracts and enhances the ability of PKA to stimulate transcription from the PEPCK promoter in cell-free reactions. The ability of okadaic acid to enhance PKA-stimulated transcription in vitro was entirely dependent on the presence of CREB in the reactions. The phospho-CREB (P-CREB) phosphatase activity present in nuclear extracts coelutes with protein Ser/Thr phosphatase type 2A (PP2A) on Mono Q, amino-hexyl Sepharose, and heparin agarose columns and was chromatographically resolved from nuclear protein Ser/Thr-phosphatase type 1 (PP1). Furthermore, P-CREB phosphatase activity in nuclear extracts was unaffected by the heat-stable protein inhibitor-2, which is a potent and selective inhibitor of PP1. Nuclear PP2A dephosphorylated P-CREB 30-fold more efficiently than did nuclear PP1. Finally, when PKA-phosphorylated CREB was treated with immunopurified PP2A and PP1, the PP2A-treated CREB did not stimulate transcription from the PEPCK promoter in vitro, whereas the PP1-treated CREB retained the ability to stimulate transcription. Nuclear PP2A appears to be the primary phosphatase that dephosphorylates PKA-phosphorylated CREB.
Topics: Amino Acid Sequence; Base Sequence; Bucladesine; Carcinoma, Hepatocellular; Cell Nucleus; Cloning, Molecular; Cyclic AMP Response Element-Binding Protein; Ethers, Cyclic; Female; Gene Expression Regulation, Neoplastic; Humans; Kinetics; Leukemia, Promyelocytic, Acute; Liver Neoplasms; Macromolecular Substances; Molecular Sequence Data; Okadaic Acid; Oligodeoxyribonucleotides; Phosphoenolpyruvate Carboxykinase (GTP); Phosphoprotein Phosphatases; Phosphorylation; Placenta; Polymerase Chain Reaction; Pregnancy; Promoter Regions, Genetic; Protein Kinases; Protein Phosphatase 2; Recombinant Proteins; Transcription, Genetic; Tumor Cells, Cultured
PubMed: 8386317
DOI: 10.1128/mcb.13.5.2822-2834.1993 -
The Journal of Biological Chemistry Nov 2000We previously reported that the activating phosphorylation on cyclin-dependent kinases in yeast (Cdc28p) and in humans (Cdk2) is removed by type 2C protein phosphatases....
We previously reported that the activating phosphorylation on cyclin-dependent kinases in yeast (Cdc28p) and in humans (Cdk2) is removed by type 2C protein phosphatases. In this study, we characterize this PP2C-like activity in HeLa cell extract and determine that it is due to PP2C beta 2, a novel PP2C beta isoform, and to PP2C alpha. PP2C alpha and PP2C beta 2 co-purified with Mg(2+)-dependent Cdk2/Cdk6 phosphatase activity in DEAE-Sepharose, Superdex-200, and Mono Q chromatographies. Moreover, purified recombinant PP2C alpha and PP2C beta 2 proteins efficiently dephosphorylated monomeric Cdk2/Cdk6 in vitro. The dephosphorylation of Cdk2 and Cdk6 by PP2C isoforms was inhibited by the binding of cyclins. We found that the PP2C-like activity in HeLa cell extract, partially purified HeLa PP2C alpha and PP2C beta 2 isoforms, and the recombinant PP2Cs exhibited a comparable substrate preference for a phosphothreonine containing substrate, consistent with the conservation of threonine residues at the site of activating phosphorylation in CDKs.
Topics: Amino Acid Sequence; Animals; Chromatography, Ion Exchange; Cyclin-Dependent Kinases; Cyclins; HeLa Cells; Humans; Isoenzymes; Mice; Molecular Sequence Data; Phosphoprotein Phosphatases; Phosphorylation; Protein Phosphatase 2; Protein Phosphatase 2C; Rats; Saccharomyces cerevisiae Proteins; Sequence Homology, Amino Acid; Substrate Specificity
PubMed: 10934208
DOI: 10.1074/jbc.M006210200