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  • Article

    Regulation of α–endosulfine, an inhibitor of protein phosphatase 2A, by multisite phosphorylation

    Author
    Mochida, Satoru
    Part of
    The FEBS journal, 2014-02, Vol.281 (4), p.1159-1169
    Abstract
    • Progression into M phase requires inhibition of heterotrimeric PP2A containing the regulatory B55 subunit (PP2A–B55) as well as the activation of cyclin‐dependent kinase 1 (Cdk1). α–endosulfine (ENSA)/cyclic AMP‐regulated 19 kDa phosphoprotein (ARPP–19) family proteins phosphorylated at S67 by Greatwall kinase bind and inhibit PP2A–B55. This study shows that endogenous kinases phosphorylate not only S67 but also two additional sites in ENSA (T28 and S109) with different kinetics at different cell‐cycle stages in Xenopus laevis intact cells and cell‐free egg extracts. When assayed in vitro, these phosphorylations had qualitatively and/or quantitatively different effects on inhibition of PP2A–B55 by ENSA. Structural analyses revealed that the most‐conserved middle region of ENSA containing S67 physically interacts with PP2A–B55 at the interface of the B55 and C subunits, where the catalytic centre of PP2A is located. As non‐phosphorylated ENSA has an intrinsic potential for PP2A–B55 inhibition, these three phosphorylations differentially affect physical interaction of the middle region of ENSA with PP2A–B55. These results suggest that the two additional phosphorylation sites together with S67 allow ENSA to function as a ‘stepwise tuner’ for PP2A–B55, which may be regulated by multiple cellular signals, rather than a simple ‘on/off’ switch. Mitosis requires both PP2A‐B55 inhibition and Cdk1 activation. This study reports that ENSA, an inhibitor of PP2A‐B55, is phosphorylated at two additional sites in addition to Ser67, the Greatwall site. These phosphorylations showed qualitatively/quantitatively different effects on ENSA's inhibition of PP2A‐B55. ENSA can be regulated by multiple cellular signals to tune PP2A‐B55 activity into several discrete levels.
    Subjects
    Animals; cell cycle; Cell Cycle - physiology; cyclin‐dependent kinase; greatwall kinase; Peptides - metabolism; Phosphorylation; protein kinase A; Protein Phosphatase 2 - metabolism; protein phosphatase 2A; Xenopus laevis
  • Article

    The crystal structure of (2a′S,2a1′S,3R,5a′S,7′R)-5-(furan-3-yl)-2a′,2a1′-dihydroxy-7′-methyldecahydro-2H-spiro[furan-3,6′-naphtho[1,8-bc]furan]-2,2′(2a′H)-dione, C19H22O7

    Authors
    Xu, Xin-Ke; Li, Can-Jie; Tan, Jin-Lin; Zhao, Hai-Yue; Lin, Qiang; Li, Yao-Lan; Zhang, Yu-Bo; Wang, Guo-Cai
    Part of
    Zeitschrift für Kristallographie. New crystal structures, 2021-12, Vol.236 (6), p.1359-1361
    Abstract
    • , orthorhombic, (no. 4),  = 6.4154(1) Å,  = 7.8782(2) Å,  = 17.1555(4) Å,  = 91.021(2)°,  = 866.93(3) Å ,  = 2, ) = 0.0326, ) = 0.1028, T = 150 K.
    Subjects
    2094891
  • Article

    SPOP is essential for DNA–protein cross-link repair in prostate cancer cells: SPOP-dependent removal of topoisomerase 2A from the topoisomerase 2A-DNA cleavage complex

    Authors
    Watanabe, Ryuta; Maekawa, Masashi; Hieda, Miki; Taguchi, Tomohiko; Miura, Noriyoshi; Kikugawa, Tadahiko; Saika, Takashi; Higashiyama, Shigeki
    Part of
    Molecular Biology of the Cell, 2020-03, Vol.31 (6), p.478-490
    Abstract
    • SPOP is a substrate recognition receptor of the CUL3 ubiquitin E3 complex and is point mutated in 10–15% of prostate cancer patients. Here, it is shown that, in prostate cancer cells, SPOP is involved in the DNA-protein cross-link repair process through the elimination of TOP2A from the TOP2A-DNA complex, which may prevent the genome instability.
    Subjects
    Cell Biology; Cell Death - drug effects; Cell Nucleus - drug effects; Cell Nucleus - metabolism; DNA Cleavage; DNA Replication - drug effects; DNA Topoisomerases, Type II - metabolism; DNA, Neoplasm - metabolism; Etoposide - pharmacology; Histones - metabolism; Humans; Hydroxyurea - pharmacology; Life Sciences & Biomedicine; Male; Models, Biological; Molecular Biology; Mutation - genetics; Nuclear Proteins - genetics; Nuclear Proteins - metabolism; Poly-ADP-Ribose Binding Proteins - metabolism; Prostatic Neoplasms - metabolism; Prostatic Neoplasms - pathology; Receptors, Androgen - metabolism; Repressor Proteins - genetics; Repressor Proteins - metabolism; Science & Technology; Topoisomerase Inhibitors - pharmacology
  • Article

    Serotonin 2A (5-HT2A) receptor affects cell-matrix adhesion and the formation and maintenance of stress fibers in HEK293 cells

    Authors
    Jayakumar, Joe Anand Kumar John; Panicker, Mitradas M.; Basu, Basudha
    Part of
    Scientific reports, 2020-12, Vol.10 (1), p.21675-21675, Article 21675
    Abstract
    • 5-HT2A, a G-protein coupled receptor, is widely expressed in the human body, including in the gastrointestinal tract, platelets and the nervous system. It mediates various functions, for e.g. learning, memory, mood regulation, platelet aggregation and vasoconstriction, but its involvement in cell-adhesion remains largely unknown. Here we report a novel role for 5-HT2A in cell-matrix adhesion.In HEK293 cells, which are loosely adherent, expression and stimulation of human or rat 5-HT2A receptor by agonists such as serotonin or 2,5-dimethoxy-4-iodoamphetamine (DOI) led to a significant increase in adhesion, while inhibition of 5-HT2A by antipsychotics, such as risperidone, olanzapine or chlorpromazine prevented it. 5-HT2A activation gave rise to stress fibers in these cells and was also required for their maintenance. Mechanistically, the 5-HT2A-mediated adhesion was mediated by downstream PKC and Rho signaling. Since 5-HT2A is associated with many disorders such as dementia, depression and schizophrenia, its role in cell-matrix adhesion could have implications for neural circuits.
    Subjects
    Amphetamines - pharmacology; Animals; Antipsychotic Agents - pharmacology; Cell-Matrix Junctions - drug effects; Cell-Matrix Junctions - genetics; Cell-Matrix Junctions - metabolism; Chlorpromazine; Chlorpromazine - pharmacology; Dementia disorders; Dopamine; Experiments; Fibers; G protein-coupled receptors; Gastrointestinal tract; HEK293 Cells; Humans; Ligands; Mental disorders; Mental Disorders - etiology; Mental Disorders - genetics; Mood; Multidisciplinary Sciences; Nervous system; Neural networks; Neuroleptics; Olanzapine; Olanzapine - pharmacology; Platelet aggregation; Protein kinase C; Psychotropic drugs; Rats; Receptor, Serotonin, 5-HT2A - physiology; Risperidone; Risperidone - pharmacology; Schizophrenia; Science & Technology; Science & Technology - Other Topics; Serotonin; Serotonin - pharmacology; Serotonin 5-HT2 Receptor Agonists - pharmacology; Serotonin 5-HT2 Receptor Antagonists - pharmacology; Serotonin S2 receptors; Stress Fibers - drug effects; Stress Fibers - genetics; Stress Fibers - metabolism; Vasoconstriction
  • Article

    High Cleavage Efficiency of a 2A Peptide Derived from Porcine Teschovirus-1 in Human Cell Lines, Zebrafish and Mice

    Authors
    Kim, Jin Hee; Lee, Sang-Rok; Li, Li-Hua; Park, Hye-Jeong; Park, Jeong-Hoh; Lee, Kwang Youl; Kim, Myeong-Kyu; Shin, Boo Ahn; Choi, Seok-Yong
    Part of
    PLoS ONE, 2011-04, Vol.6 (4), p.e18556--e18556
    Abstract
    • When expression of more than one gene is required in cells, bicistronic or multicistronic expression vectors have been used. Among various strategies employed to construct bicistronic or multicistronic vectors, an internal ribosomal entry site (IRES) has been widely used. Due to the large size and difference in expression levels between genes before and after IRES, however, a new strategy was required to replace IRES. A self-cleaving 2A peptide could be a good candidate to replace IRES because of its small size and high cleavage efficiency between genes upstream and downstream of the 2A peptide. Despite the advantages of the 2A peptides, its use is not widespread because (i) there are no publicly available cloning vectors harboring a 2A peptide gene and (ii) comprehensive comparison of cleavage efficiency among various 2A peptides reported to date has not been performed in different contexts. Here, we generated four expression plasmids each harboring different 2A peptides derived from the foot-and-mouth disease virus, equine rhinitis A virus, Thosea asigna virus and porcine teschovirus-1, respectively, and evaluated their cleavage efficiency in three commonly used human cell lines, zebrafish embryos and adult mice. Western blotting and confocal microscopic analyses revealed that among the four 2As, the one derived from porcine teschovirus-1 (P2A) has the highest cleavage efficiency in all the contexts examined. We anticipate that the 2A-harboring cloning vectors we generated and the highest efficiency of the P2A peptide we demonstrated would help biomedical researchers easily adopt the 2A technology when bicistronic or multicistronic expression is required.
    Subjects
    Amino Acid Sequence; Amino acids; Analysis; Animals; Base Sequence; Biology; Biomedical research; Biotechnology; Cell Line; Cell lines; Cells; Cells (Biology); Chemistry; Cleavage; Cloning; Cloning vectors; Danio rerio; Deoxyribonucleic acid; DNA; Efficiency; Embryo, Nonmammalian - metabolism; Embryos; Expression vectors; Foot & mouth disease; Foot-and-mouth disease; Gene Expression; Genes; Genetic engineering; Growth factors; Humans; Liver - metabolism; Localization; Medical research; Medical schools; Mice; Molecular Sequence Data; Multidisciplinary; Muridae; Nematostella vectensis; Peptides; Peptides - chemistry; Peptides - genetics; Peptides - metabolism; Plasmids; Plasmids - genetics; Proteins; Rhinitis; Sus scrofa - virology; Teschovirus - metabolism; Viral Proteins - chemistry; Viral Proteins - genetics; Viral Proteins - metabolism; Viruses; Western blotting; Zebrafish; Zebrafish - embryology; Zebrafish - genetics; Zebrafish - metabolism
  • Article

    Altered interplay between endoplasmic reticulum and mitochondria in Charcot–Marie–Tooth type 2A neuropathy

    Authors
    Bernard-Marissal, Nathalie; van Hameren, Gerben; Juneja, Manisha; Pellegrino, Christophe; Louhivuori, Lauri; Bartesaghi, Luca; Rochat, Cylia; El Mansour, Omar; Médard, Jean-Jacques; Croisier, Marie; Maclachlan, Catherine; Poirot, Olivier; Uhlén, Per; Timmerman, Vincent; Tricaud, Nicolas; Schneider, Bernard L.; Chrast, Roman
    Part of
    Proceedings of the National Academy of Sciences, 2019-02, Vol.116 (6), p.2328-2337
    Abstract
    • SignificanceInteractions between mitochondria and the endoplasmic reticulum (ER) at the level of mitochondria-associated membranes (MAM) constitute a key signaling hub, emerging as a shared target altered in multiple neurodegenerative diseases. We use both in vivo and in vitro models of Charcot–Marie–Tooth type 2A, an axonal form of neuropathy, to demonstrate that the presence of mutated Mitofusin 2 leads to altered MAM. In neurons, these modifications occur concomitantly with activation of ER stress response, dysregulated calcium handling, and alterations in mitochondrial morphology and transport, collectively contributing to axonopathy. Importantly, the reported results indicate that the pathological consequences of mutated Mitofusin 2 may be targeted with drugs reinforcing the ER–mitochondria cross-talk and/or reducing ER stress.
    Subjects
    Animals; Axonal transport; Axons - metabolism; Biological Sciences; Biological Transport; Calcium; Calcium (reticular); Charcot-Marie-Tooth disease; Charcot-Marie-Tooth Disease - genetics; Charcot-Marie-Tooth Disease - metabolism; Charcot-Marie-Tooth Disease - physiopathology; Crosstalk; Defects; Degeneration; Disease Models, Animal; Drug therapy; Endoplasmic reticulum; Endoplasmic Reticulum - metabolism; Endoplasmic Reticulum - ultrastructure; Female; Fibroblasts; Gait; Life Sciences; Locomotion - genetics; Male; Mathematical morphology; Medicin och hälsovetenskap; Membranes; Mice; Mice, Transgenic; Mitochondria; Mitochondria - metabolism; Mitochondria - ultrastructure; Motor neurons; Motor Neurons - metabolism; Multidisciplinary; Muscle Denervation; Muscle Fibers, Slow-Twitch; Mutation; Neurodegeneration; Neurons and Cognition; Peripheral neuropathy; Pharmacology; PNAS Plus; Proteins; Signal Transduction
  • Article

    Allosteric Activators of Protein Phosphatase 2A Display Broad Antitumor Activity Mediated by Dephosphorylation of MYBL2 (Retracted article. See vol. 185, pg. 3058, 2022)

    Authors
    Morita, Ken; He, Shuning; Nowak, Radoslaw P.; Wang, Jinhua; Zimmerman, Mark W.; Fu, Cong; Durbin, Adam D.; Martel, Megan W.; Prutsch, Nicole; Gray, Nathanael S.; Fischer, Eric S.; Look, A. Thomas
    Part of
    Cell, 2020-04, Vol.181 (3), p.702-715.e20
    Abstract
    • Protein phosphatase 2A (PP2A) enzymes can suppress tumors, but they are often inactivated in human cancers overexpressing inhibitory proteins. Here, we identify a class of small-molecule iHAPs (improved heterocyclic activators of PP2A) that kill leukemia cells by allosterically assembling a specific heterotrimeric PP2A holoenzyme consisting of PPP2R1A (scaffold), PPP2R5E (B56 epsilon, regulatory), and PPP2CA (catalytic) subunits. One compound, iHAP1, activates this complex but does not inhibit dopamine receptor D2, a mediator of neurologic toxicity induced by perphenazine and related neuroleptics. The PP2A complex activated by iHAP1 dephosphorylates the MYBL2 transcription factor on Ser241, causing irreversible arrest of leukemia and other cancer cells in prometaphase. In contrast, SMAPs, a separate class of compounds, activate PP2A holoenzymes containing a different regulatory subunit, do not dephosphorylate MYBL2, and arrest tumor cells in G1 phase. Our findings demonstrate that small molecules can serve as allosteric switches to activate distinct PP2A complexes with unique substrates.
    Subjects
    Biochemistry & Molecular Biology; Cell Biology; Life Sciences & Biomedicine; Science & Technology
  • Article

    Systematic comparison of 2A peptides for cloning multi-genes in a polycistronic vector

    Authors
    Liu, Ziqing; Chen, Olivia; Wall, J Blake Joseph; Zheng, Michael; Zhou, Yang; Wang, Li; Ruth Vaseghi, Haley; Qian, Li; Liu, Jiandong
    Part of
    Scientific Reports, 2017-05, Vol.7 (1), p.2193--9, Article 2193
    Abstract
    • AbstractCloning of multiple genes in a single vector has greatly facilitated both basic and translational studies that require co-expression of multiple factors or multi-units of complex protein. Many strategies have been adopted, among which 2A “self-cleaving” peptides have garnered increased interest for their polycistronic nature, small size and high “cleavage” efficiency. However, broad application of 2 A peptides is limited by the lack of systematic comparison of different 2As alone or in combination. Here we characterized the effect of varying gene position and 2As on the expression of proteins encoded in bi-, tri-, or quad-cistronic constructs. Using direct cardiac reprogramming as an example, we further determined the effect of varied 2As on the efficiency of fluorescent cell labeling and cell fate conversion. We found that the expression of fluorophores decreased as it was moved towards the end of the construct while reprogramming was most efficient with the fluorophore at the second position. Moreover, quad-cistronic TPE2A constructs resulted in more efficient reprogramming than 3P2A or PTE2A constructs. We expect that the bi-, tri-, and quad-cistronic vectors constructed here and our results on protein expression ratios from different 2A constructs could serve to guide future utilization of 2A peptides in basic research and clinical applications.
    Subjects
    Animals; Cell fate; Cell Line; Cell Tracking - methods; Cellular Reprogramming Techniques; Cloning; Cloning vectors; Expression vectors; Fluorophores; Gene Expression; Gene Order; Genetic Vectors - chemistry; Genetic Vectors - genetics; Heart diseases; Humans; Mice; Multidisciplinary; Peptides; Peptides - genetics; Promoter Regions, Genetic; Tandem Repeat Sequences; Therapeutic applications
  • Article

    Polymorphism in the serotonin receptor 2a (HTR2A) gene as possible predisposal factor for aggressive traits

    Authors
    Banlaki, Zsofia; Elek, Zsuzsanna; Nanasi, Tibor; Szekely, Anna; Nemoda, Zsofia; Sasvari-Szekely, Maria; Ronai, Zsolt
    Part of
    PloS one, 2015-02, Vol.10 (2), p.e0117792-e0117792
    Abstract
    • Aggressive manifestations and their consequences are a major issue of mankind, highlighting the need for understanding the contributory factors. Still, aggression-related genetic analyses have so far mainly been conducted on small population subsets such as individuals suffering from a certain psychiatric disorder or a narrow-range age cohort, but no data on the general population is yet available. In the present study, our aim was to identify polymorphisms in genes affecting neurobiological processes that might explain some of the inter-individual variation between aggression levels in the non-clinical Caucasian adult population. 55 single nucleotide polymorphisms (SNP) were simultaneously determined in 887 subjects who also filled out the self-report Buss-Perry Aggression Questionnaire (BPAQ). Single marker association analyses between genotypes and aggression scores indicated a significant role of rs7322347 located in the HTR2A gene encoding serotonin receptor 2a following Bonferroni correction for multiple testing (p = 0.0007) both for males and females. Taking the four BPAQ subscales individually, scores for Hostility, Anger and Physical Aggression showed significant association with rs7322347 T allele in themselves, while no association was found with Verbal Aggression. Of the subscales, relationship with rs7322347 was strongest in the case of Hostility, where statistical significance virtually equaled that observed with the whole BPAQ. In conclusion, this is the first study to our knowledge analyzing SNPs in a wide variety of genes in terms of aggression in a large sample-size non-clinical adult population, also describing a novel candidate polymorphism as predisposal to aggressive traits.
    Subjects
    Adolescent; Adult; Aged; Aggression; Aggression - physiology; Aggressive behavior; Aggressiveness; Alleles; Analysis; Associations; Behavior; Binding sites; Deoxyribonucleic acid; DNA; Female; Females; Gene expression; Gene polymorphism; Genes; Genetic analysis; Genetic aspects; Genetic Association Studies; Genetic testing; Genotype; Genotypes; Hostility; Humans; Male; Males; Medical research; Mental disorders; Mental illness; MicroRNAs; Middle Aged; Molecular biology; Phenols; Phenols (Class of compounds); Phenotype; Polymorphism; Polymorphism, Single Nucleotide; Population (statistical); Receptor, Serotonin, 5-HT2A - genetics; Schizophrenia; Self Report; Serotonin; Signal transduction; Single nucleotide polymorphisms; Single-nucleotide polymorphism; Studies; Young Adult
  • Article

    Integrator Recruits Protein Phosphatase 2A to Prevent Pause Release and Facilitate Transcription Termination

    Authors
    Huang, Kai-Lieh; Jee, David; Stein, Chad B.; Elrod, Nathan D.; Henriques, Telmo; Mascibroda, Lauren G.; Baillat, David; Russell, William K.; Adelman, Karen; Wagner, Eric J.
    Part of
    Molecular Cell, 2020-10, Vol.80 (2), p.345-358.e9
    Abstract
    • Efficient release of promoter-proximally paused RNA Pol II into productive elongation is essential for gene expression. Recently, we reported that the Integrator complex can bind paused RNA Pol II and drive premature transcription termination, potently attenuating the activity of target genes. Premature termination requires RNA cleavage by the endonuclease subunit of Integrator, but the roles of other Integrator subunits in gene regulation have yet to be elucidated. Here we report that Integrator subunit 8 (IntS8) is critical for transcription repression and required for association with protein phosphatase 2A (PP2A). We find that Integrator-bound PP2A dephosphorylates the RNA Pol II C-terminal domain and Spt5, preventing the transition to productive elongation. Thus, blocking PP2A association with Integrator stimulates pause release and gene activity. These results reveal a second catalytic function associated with Integrator-mediated transcription termination and indicate that control of productive elongation involves active competition between transcriptional kinases and phosphatases. [Display omitted]
      • A short motif in IntS8 mediates association with protein phosphatase 2A (PP2A)
      • Recruitment of PP2A is necessary for Integrator-mediated gene repression
      • Integrator-bound PP2A dephosphorylates residues within the RNA Pol II CTD and Spt5
      • PP2A antagonizes transcriptional kinases to prevent productive elongation The Integrator complex binds promoter-proximally paused RNA Pol II and drives premature transcription termination. Huang et al. identified a motif within Integrator subunit 8 (INTS8) that recruits protein phosphatase 2A (PP2A) to RNA Pol II. Integrator-PP2A then dephosphorylates residues within Spt5 and the RNA Pol II CTD to inhibit pause release and facilitate termination.
    Subjects
    Amino Acid Motifs; Amino Acid Sequence; Animals; Biochemistry & Molecular Biology; Cell Biology; Conserved Sequence; Drosophila melanogaster; Drosophila Proteins - chemistry; Drosophila Proteins - metabolism; Gene Expression Regulation; Genetic Loci; Humans; Integrator; Life Sciences & Biomedicine; Molecular Biology; paused RNA Pol II; phosphatase; Phosphorylation; Promoter Regions, Genetic; Protein Phosphatase 2 - metabolism; Protein Subunits - chemistry; Protein Subunits - metabolism; RNA Polymerase II - chemistry; RNA Polymerase II - metabolism; Science & Technology; Signal Transduction; Substrate Specificity; Transcription Factors - chemistry; Transcription Factors - metabolism; transcription regulation; transcription termination; Transcription Termination, Genetic
  • Article

    Epstein-Barr Virus Latent Membrane Protein 2A (LMP2A) Enhances ATP Production in B Cell Tumors through mTOR and HIF-1α

    Authors
    Incrocci, Ryan; Monroy Del Toro, Rosalinda; Devitt, Grace; Salimian, Melody; Braich, Kamaljit; Swanson-Mungerson, Michelle
    Part of
    International journal of molecular sciences, 2024-04, Vol.25 (7), p.3944
    Abstract
    • Epstein-Barr Virus (EBV) exists in a latent state in 90% of the world's population and is linked to numerous cancers, such as Burkitt's Lymphoma, Hodgkin's, and non-Hodgkin's Lymphoma. One EBV latency protein, latency membrane protein 2A (LMP2A), is expressed in multiple latency phenotypes. LMP2A signaling has been extensively studied and one target of LMP2A is the mammalian target of rapamycin (mTOR). Since mTOR has been linked to reprogramming tumor metabolism and increasing levels of hypoxia-inducible factor 1 α (HIF-1α), we hypothesized that LMP2A would increase HIF-1α levels to enhance ATP generation in B lymphoma cell lines. Our data indicate that LMP2A increases ATP generation in multiple Burkitt lymphoma cell lines that were dependent on HIF-1α. Subsequent studies indicate that the addition of the mTOR inhibitor, rapamycin, blocked the LMP2A-dependent increase in HIF-1α. Further studies demonstrate that LMP2A does not increase HIF-1α levels by increasing HIF-1α RNA or STAT3 activation. In contrast, LMP2A and mTOR-dependent increase in HIF-1α required mTOR-dependent phosphorylation of p70 S6 Kinase and 4E-BP1. These findings implicate the importance of LMP2A in promoting B cell lymphoma survival by increasing ATP generation and identifying potential pharmaceutical targets to treat EBV-associated tumors.
    Subjects
    Adenosine Triphosphate; B cells; Biotechnology industry; Burkitt Lymphoma; Cell cycle; Epstein-Barr virus; Epstein-Barr Virus Infections; Flow cytometry; Herpesvirus 4, Human; Humans; Infections; International economic relations; Kinases; latency membrane protein 2A (LMP2A); Lymphoma; mammalian target of rapamycin (mTOR); Membrane Proteins; metabolism; Non-Hodgkin's lymphomas; Phosphorylation; Protein synthesis; Proteins; RNA; Scientific equipment and supplies industry; Statistical analysis; TOR Serine-Threonine Kinases; Tumors
  • Article

    Phase 2a study of ataluren-mediated dystrophin production in patients with nonsense mutation Duchenne muscular dystrophy

    Authors
    Finkel, Richard S; Flanigan, Kevin M; Wong, Brenda; Bönnemann, Carsten; Sampson, Jacinda; Sweeney, H Lee; Reha, Allen; Northcutt, Valerie J; Elfring, Gary; Barth, Jay; Peltz, Stuart W
    Part of
    PloS one, 2013-12, Vol.8 (12), p.e81302-e81302
    Abstract
    • Approximately 13% of boys with Duchenne muscular dystrophy (DMD) have a nonsense mutation in the dystrophin gene, resulting in a premature stop codon in the corresponding mRNA and failure to generate a functional protein. Ataluren (PTC124) enables ribosomal readthrough of premature stop codons, leading to production of full-length, functional proteins. This Phase 2a open-label, sequential dose-ranging trial recruited 38 boys with nonsense mutation DMD. The first cohort (n = 6) received ataluren three times per day at morning, midday, and evening doses of 4, 4, and 8 mg/kg; the second cohort (n = 20) was dosed at 10, 10, 20 mg/kg; and the third cohort (n = 12) was dosed at 20, 20, 40 mg/kg. Treatment duration was 28 days. Change in full-length dystrophin expression, as assessed by immunostaining in pre- and post-treatment muscle biopsy specimens, was the primary endpoint. Twenty three of 38 (61%) subjects demonstrated increases in post-treatment dystrophin expression in a quantitative analysis assessing the ratio of dystrophin/spectrin. A qualitative analysis also showed positive changes in dystrophin expression. Expression was not associated with nonsense mutation type or exon location. Ataluren trough plasma concentrations active in the mdx mouse model were consistently achieved at the mid- and high- dose levels in participants. Ataluren was generally well tolerated. Ataluren showed activity and safety in this short-term study, supporting evaluation of ataluren 10, 10, 20 mg/kg and 20, 20, 40 mg/kg in a Phase 2b, double-blind, long-term study in nonsense mutation DMD. ClinicalTrials.gov NCT00264888.
    Subjects
    Analysis; Biopsy; Child; Clinical trials; Codon; Codon, Nonsense - genetics; Codons; Drug dosages; Drug therapy; Duchenne muscular dystrophy; Duchenne's muscular dystrophy; Dystrophin; Dystrophin - metabolism; Dystrophy; Genetic aspects; Hospitals; Humans; Kinases; Male; Medicine; mRNA; Muscles; Muscular dystrophy; Muscular Dystrophy, Duchenne - drug therapy; Muscular Dystrophy, Duchenne - metabolism; Mutation; Nonsense mutation; Oxadiazoles - therapeutic use; Proteins; Qualitative analysis; Quantitative analysis; Rodents; Spectrin; Stop codon; Utrophin
  • Article

    Structure of the Protein Phosphatase 2A Holoenzyme

    Authors
    Xu, Yanhui; Xing, Yongna; Chen, Yu; Chao, Yang; Lin, Zheng; Fan, Eugene; Yu, Jong W.; Strack, Stefan; Jeffrey, Philip D.; Shi, Yigong; Brookhaven National Laboratory (BNL) National Synchrotron Light Source
    Part of
    Cell, 2006-12, Vol.127 (6), p.1239-1251
    Abstract
    • Protein Phosphatase 2A (PP2A) plays an essential role in many aspects of cellular physiology. The PP2A holoenzyme consists of a heterodimeric core enzyme, which comprises a scaffolding subunit and a catalytic subunit, and a variable regulatory subunit. Here we report the crystal structure of the heterotrimeric PP2A holoenzyme involving the regulatory subunit B′/B56/PR61. Surprisingly, the B′/PR61 subunit has a HEAT-like ( huntingtin- elongation- A subunit- TOR-like) repeat structure, similar to that of the scaffolding subunit. The regulatory B′/B56/PR61 subunit simultaneously interacts with the catalytic subunit as well as the conserved ridge of the scaffolding subunit. The carboxyterminus of the catalytic subunit recognizes a surface groove at the interface between the B′/B56/PR61 subunit and the scaffolding subunit. Compared to the scaffolding subunit in the PP2A core enzyme, formation of the holoenzyme forces the scaffolding subunit to undergo pronounced conformational rearrangements. This structure reveals significant ramifications for understanding the function and regulation of PP2A.
    Subjects
    Amino Acid Sequence; Catalytic Domain; CRYSTAL STRUCTURE; Crystallography, X-Ray; FUNCTIONS; General Biochemistry, Genetics and Molecular Biology; Holoenzymes - chemistry; Holoenzymes - metabolism; Humans; INTERFACES; MATERIALS SCIENCE; Methylation; Models, Molecular; Molecular Sequence Data; Multiprotein Complexes - metabolism; national synchrotron light source; PHOSPHATASES; Phosphoprotein Phosphatases - chemistry; Phosphoprotein Phosphatases - metabolism; PHYSIOLOGY; Protein Conformation; Protein Phosphatase 2; Protein Structure, Tertiary; PROTEINS; REGULATIONS; Sequence Alignment; Software; SURFACES
  • Article

    The biogenesis of active protein phosphatase 2A holoenzymes: a tightly regulated process creating phosphatase specificity

    Authors
    Sents, Ward; Ivanova, Elitsa; Lambrecht, Caroline; Haesen, Dorien; Janssens, Veerle
    Part of
    The FEBS Journal, 2013-01, Vol.280 (2), p.644-661
    Abstract
    • Protein phosphatase type 2A (PP2A) enzymes constitute a large family of Ser/Thr phosphatases with multiple functions in cellular signaling and physiology. The composition of heterotrimeric PP2A holoenzymes, resulting from the combinatorial assembly of a catalytic C subunit, a structural A subunit, and regulatory B‐type subunit, provides the essential determinants for substrate specificity, subcellular targeting, and fine‐tuning of phosphatase activity, largely explaining why PP2A is functionally involved in so many diverse physiological processes, sometimes in seemingly opposing ways. In this review, we highlight how PP2A holoenzyme biogenesis and enzymatic activity are controlled by a sophisticatedly coordinated network of five PP2A modulators, consisting of α4, phosphatase 2A phosphatase activator (PTPA), leucine carboxyl methyl transferase 1 (LCMT1), PP2A methyl esterase 1 (PME‐1) and, potentially, target of rapamycin signaling pathway regulator‐like 1 (TIPRL1), which serve to prevent promiscuous phosphatase activity until the holoenzyme is completely assembled. Likewise, these modulators may come into play when PP2A holoenzymes are disassembled following particular cellular stresses. Malfunctioning of these cellular control mechanisms contributes to human disease. The potential therapeutic benefits or pitfalls of interfering with these regulatory mechanisms will be briefly discussed.
    Subjects
    Adaptor Proteins, Signal Transducing; Biochemistry; Carboxylic Ester Hydrolases - metabolism; carboxymethylation; Cell Biology; Holoenzymes - biosynthesis; Holoenzymes - metabolism; Humans; Intracellular Signaling Peptides and Proteins - metabolism; leucine carboxyl methyl transferase 1 (LCMT1); Models, Biological; Molecular Biology; Molecular Chaperones; peptidyl‐prolyl cis/trans isomerase; phosphatase 2A phosphatase activator (PTPA); Phosphoprotein Phosphatases - metabolism; Protein O-Methyltransferase - metabolism; Protein Phosphatase 2 - biosynthesis; Protein Phosphatase 2 - metabolism; protein phosphatase type 2A (PP2A); protein phosphatase type 2A methyl esterase 1 (PME‐1); protein phosphatase type 4 (PP4); protein phosphatase type 6 (PP6); Substrate Specificity; target of rapamycin signaling pathway regulator‐like 1 (TIPRL1)
  • Article

    Presynaptic serotonin 2A receptors modulate thalamocortical plasticity and associative learning

    Authors
    Barre, Alexander; Berthoux, Coralie; De Bundel, Dimitri; Valjent, Emmanuel; Bockaert, Joël; Marin, Philippe; Bécamel, Carine
    Part of
    Proceedings of the National Academy of Sciences - PNAS, 2016-03, Vol.113 (10), p.E1382-E1391
    Abstract
    • Higher-level cognitive processes strongly depend on a complex interplay between mediodorsal thalamus nuclei and the prefrontal cortex (PFC). Alteration of thalamofrontal connectivity has been involved in cognitive deficits of schizophrenia. Prefrontal serotonin (5-HT)2A receptors play an essential role in cortical network activity, but the mechanism underlying their modulation of glutamatergic transmission and plasticity at thalamocortical synapses remains largely unexplored. Here, we show that 5-HT2A receptor activation enhances NMDA transmission and gates the induction of temporal-dependent plasticity mediated by NMDA receptors at thalamocortical synapses in acute PFC slices. Expressing 5-HT2A receptors in the mediodorsal thalamus (presynaptic site) of 5-HT2A receptor-deficient mice, but not in the PFC (postsynaptic site), using a viral gene-delivery approach, rescued the otherwise absent potentiation of NMDA transmission, induction of temporal plasticity, and deficit in associative memory. These results provide, to our knowledge, the first physiological evidence of a role of presynaptic 5-HT2A receptors located at thalamocortical synapses in the control of thalamofrontal connectivity and the associated cognitive functions.
    Subjects
    Animal cognition; Animals; Association Learning - physiology; Biological Sciences; Blotting, Western; Brain; Cerebral Cortex - metabolism; Cerebral Cortex - physiology; Electrophysiological Phenomena; Life Sciences; Mice, Inbred C57BL; Mice, Knockout; Microscopy, Fluorescence; Neurology; Neuronal Plasticity - genetics; Neuronal Plasticity - physiology; Neuropsychology; PNAS Plus; Prefrontal Cortex - metabolism; Prefrontal Cortex - physiology; Protein Kinase C - metabolism; Rats, Sprague-Dawley; Receptor, Serotonin, 5-HT2A - genetics; Receptor, Serotonin, 5-HT2A - metabolism; Receptor, Serotonin, 5-HT2A - physiology; Receptors, N-Methyl-D-Aspartate - metabolism; Receptors, N-Methyl-D-Aspartate - physiology; Rodents; Schizophrenia; Serotonin; Synapses - metabolism; Synapses - physiology; Synaptic Transmission - genetics; Synaptic Transmission - physiology; Thalamus - metabolism; Thalamus - physiology; Type C Phospholipases - metabolism
  • Article

    Phase 1/2a Study of the Malaria Vaccine Candidate Apical Membrane Antigen-1 (AMA-1) Administered in Adjuvant System AS01B or AS02A

    Authors
    Spring, Michele D; Cummings, James F; Ockenhouse, Christian F; Dutta, Sheetij; Reidler, Randall; Angov, Evelina; Bergmann-Leitner, Elke; Stewart, V Ann; Bittner, Stacey; Juompan, Laure; Kortepeter, Mark G; Nielsen, Robin; Krzych, Urszula; Tierney, Ev; Ware, Lisa A; Dowler, Megan; Hermsen, Cornelus C; Sauerwein, Robert W; de Vlas, Sake J; Ofori-Anyinam, Opokua; Lanar, David E; Williams, Jack L; Kester, Kent E; Tucker, Kathryn; Shi, Meng; Malkin, Elissa; Long, Carole; Diggs, Carter L; Soisson, Lorraine; Dubois, Marie-Claude; Ballou, W Ripley; Cohen, Joe; Heppner, Jr, D Gray
    Part of
    PLoS ONE, 2009-04, Vol.4 (4), p.e5254--e5254
    Abstract
    • This Phase 1/2a study evaluated the safety, immunogenicity, and efficacy of an experimental malaria vaccine comprised of the recombinant Plasmodium falciparum protein apical membrane antigen-1 (AMA-1) representing the 3D7 allele formulated with either the AS01B or AS02A Adjuvant Systems. After a preliminary safety evaluation of low dose AMA-1/AS01B (10 microg/0.5 mL) in 5 adults, 30 malaria-naïve adults were randomly allocated to receive full dose (50 microg/0.5 mL) of AMA-1/AS01B (n = 15) or AMA-1/AS02A (n = 15), followed by a malaria challenge. All vaccinations were administered intramuscularly on a 0-, 1-, 2-month schedule. All volunteers experienced transient injection site erythema, swelling and pain. Two weeks post-third vaccination, anti-AMA-1 Geometric Mean Antibody Concentrations (GMCs) with 95% Confidence Intervals (CIs) were high: low dose AMA-1/AS01B 196 microg/mL (103-371 microg/mL), full dose AMA-1/AS01B 279 microg/mL (210-369 microg/mL) and full dose AMA-1/AS02A 216 microg/mL (169-276 microg/mL) with no significant difference among the 3 groups. The three vaccine formulations elicited equivalent functional antibody responses, as measured by growth inhibition assay (GIA), against homologous but not against heterologous (FVO) parasites as well as demonstrable interferon-gamma (IFN-gamma) responses. To assess efficacy, volunteers were challenged with P. falciparum-infected mosquitoes, and all became parasitemic, with no significant difference in the prepatent period by either light microscopy or quantitative polymerase chain reaction (qPCR). However, a small but significant reduction of parasitemia in the AMA-1/AS02A group was seen with a statistical model employing qPCR measurements. All three vaccine formulations were found to be safe and highly immunogenic. These immune responses did not translate into significant vaccine efficacy in malaria-naïve adults employing a primary sporozoite challenge model, but encouragingly, estimation of parasite growth rates from qPCR data may suggest a partial biological effect of the vaccine. Further evaluation of the immunogenicity and efficacy of the AMA-1/AS02A formulation is ongoing in a malaria-experienced pediatric population in Mali. www.clinicaltrials.gov NCT00385047.
    Subjects
    Adjuvants, Immunologic - administration & dosage; Adjuvants, Immunologic - pharmacology; Adolescent; Adult; Adults; Alleles; Analysis; Animals; Antigens; Antigens, Protozoan - administration & dosage; Antigens, Protozoan - genetics; Antigens, Protozoan - immunology; Appropriate technology; Aquatic insects; Biological effects; Biological response modifiers; Confidence intervals; Double-Blind Method; Drug Combinations; Effectiveness; Enzyme-Linked Immunosorbent Assay; Enzymes; Erythema; Evaluation; Formulations; Goats; Growth rate; Health care; Homology; Humans; Immune response; Immunogenicity; Immunology; Infectious Diseases; Interferon; Light microscopy; Lipid A - administration & dosage; Lipid A - analogs & derivatives; Lipid A - pharmacology; Lymphocytes; Malaria; Malaria vaccine; Malaria Vaccines - administration & dosage; Malaria Vaccines - adverse effects; Malaria Vaccines - immunology; Malaria, Falciparum - prevention & control; Mathematical models; Medical research; Medical societies; Medicine, Experimental; Membrane proteins; Membrane Proteins - administration & dosage; Membrane Proteins - genetics; Membrane Proteins - immunology; Microbiology; Middle Aged; Mosquitoes; Multidisciplinary; Pain; Parasitemia; Parasites; Peptides; Plasmodium falciparum; Plasmodium falciparum - immunology; Plasmodium falciparum - metabolism; Polymerase chain reaction; Population (statistical); Proteins; Protozoan Proteins - administration & dosage; Protozoan Proteins - genetics; Protozoan Proteins - immunology; Safety; Saponins - administration & dosage; Saponins - pharmacology; Statistical analysis; Studies; Vaccination; Vaccine efficacy; Vaccines; Vector-borne diseases; γ-Interferon
  • Article

    B′-protein phosphatase 2A is a functional binding partner of delta-retroviral integrase

    Author
    Maertens, Goedele N
    Part of
    Nucleic Acids Research, 2016-01, Vol.44 (1), p.364-376
    Abstract
    • To establish infection, a retrovirus must insert a DNA copy of its RNA genome into host chromatin. This reaction is catalysed by the virally encoded enzyme integrase (IN) and is facilitated by viral genus-specific host factors. Herein, cellular serine/threonine protein phosphatase 2A (PP2A) is identified as a functional IN binding partner exclusive to δ-retroviruses, including human T cell lymphotropic virus type 1 and 2 (HTLV-1 and HTLV-2) and bovine leukaemia virus (BLV). PP2A is a heterotrimer composed of a scaffold, catalytic and one of any of four families of regulatory subunits, and the interaction is specific to the B' family of the regulatory subunits. B'-PP2A and HTLV-1 IN display nuclear co-localization, and the B' subunit stimulates concerted strand transfer activity of δ-retroviral INs in vitro. The protein-protein interaction interface maps to a patch of highly conserved residues on B', which when mutated render B' incapable of binding to and stimulating HTLV-1 and -2 IN strand transfer activity.
    Subjects
    Amino Acid Sequence; Animals; Carrier Proteins - metabolism; Cattle; Cell Line; Deltaretrovirus - enzymology; Deltaretrovirus - metabolism; Enzyme Activation; Genetics; Human T-lymphotropic virus 1; Human T-lymphotropic virus 1 - enzymology; Human T-lymphotropic virus 1 - metabolism; Human T-lymphotropic virus 2; Humans; Integrases - metabolism; Leukemia Virus, Bovine - enzymology; Leukemia Virus, Bovine - metabolism; Models, Molecular; Molecular Sequence Data; Nucleic Acid Enzymes; Protein Binding; Protein Conformation; Protein Interaction Domains and Motifs; Protein Interaction Mapping; Protein Phosphatase 2 - chemistry; Protein Phosphatase 2 - metabolism; Protein Subunits; Sequence Alignment; Virus Integration
  • Article

    Protein phosphatase 2A–mediated flotillin-1 dephosphorylation up-regulates endothelial cell migration and angiogenesis regulation

    Authors
    Thalwieser, Zsófia; Király, Nikolett; Fonódi, Márton; Csortos, Csilla; Boratkó, Anita
    Part of
    The Journal of biological chemistry, 2019-12, Vol.294 (52), p.20196-20206
    Abstract
    • Endothelial cells have key functions in endothelial barrier integrity and in responses to angiogenic signals that promote cell proliferation, cell migration, cytoskeletal reorganization, and formation of new blood vessels. These functions highly depend on protein–protein interactions in cell–cell junction and cell attachment complexes and on interactions with cytoskeletal proteins. Protein phosphatase 2A (PP2A) dephosphorylates several target proteins involved in cytoskeletal dynamics and cell adhesion. Our goal was to find new interacting and substrate proteins of the PP2A-B55α holoenzyme in bovine pulmonary endothelial cells. Using LC-MS/MS analysis, we identified flotillin-1 as a protein that binds recombinant GSH S-transferase–tagged PP2A-B55α. Immunoprecipitation experiments, proximity ligation assays, and immunofluorescent staining confirmed the interaction between these two endogenous proteins in endothelial cells. Originally, flotillins were described as regulatory proteins for axon regeneration, but they appear to function in many cellular processes, such as membrane receptor signaling, endocytosis, and cell adhesion. Ser315 is a known PKC-targeted site in flotillin-1. Utilizing phosphomutants of flotillin-1 and the NanoBiT luciferase assay, we show here that phosphorylation/dephosphorylation of Ser315 in flotillin-1 significantly affects its interaction with PP2A-B55α and that PP2A-B55α dephosphorylates phospho-Ser315. Spreading, attachment, migration, and in vitro tube formation rates of S315A variant–overexpressing cells were faster than those of nontransfected or S315D-transfected cells. These results indicate that the PP2A–flotillin-1 interaction identified here affects major physiological activities of pulmonary endothelial cells.
    Subjects
    angiogenesis; Animals; Carbazoles - pharmacology; Cattle; Cell Biology; cell migration; Cell Movement; Cells, Cultured; endothelial cell; Endothelial Cells - cytology; Endothelial Cells - metabolism; Holoenzymes - metabolism; Membrane Proteins - genetics; Membrane Proteins - metabolism; Mutagenesis, Site-Directed; Neovascularization, Physiologic; phosphorylation; Phosphorylation - drug effects; Protein Interaction Domains and Motifs; Protein Kinase C - metabolism; protein phosphatase 2 (PP2A); Protein Phosphatase 2 - antagonists & inhibitors; Protein Phosphatase 2 - genetics; Protein Phosphatase 2 - metabolism; Protein Subunits - genetics; Protein Subunits - metabolism; RNA Interference; RNA, Small Interfering - metabolism; Up-Regulation
  • Article

    Protein phosphatase 2A stimulates activation of TFEB and TFE3 transcription factors in response to oxidative stress

    Authors
    Martina, José A.; Puertollano, Rosa
    Part of
    Journal of Biological Chemistry, 2018-08, Vol.293 (32), p.12525-12534
    Abstract
    • Adaptations and responses to stress conditions are fundamental processes that all cells must accomplish to maintain or restore cellular homeostasis. Cells have a plethora of response pathways to mitigate the effect of different environmental stressors. The transcriptional regulators transcription factor EB (TFEB) and transcription factor binding to IGHM enhancer 3 (TFE3) play a key role in the control of these stress pathways. Therefore, understanding their regulation under different stress conditions is of great interest. Here, using a range of human and murine cells, we show that TFEB and TFE3 are activated upon induction of acute oxidative stress by sodium arsenite via an mTOR complex 1 (mTORC1)-independent process. We found that the mechanism of arsenite-stimulated TFEB and TFE3 activation instead involves protein phosphatase 2A (PP2A)-mediated dephosphorylation at Ser-211 and Ser-321, respectively. Depletion of either the catalytic (PPP2CA+B) or regulatory (PPP2R2A/B55α) subunits of PP2A, as well as PP2A inactivation with the specific inhibitor okadaic acid, abolished TFEB and TFE3 activation in response to sodium arsenite. Conversely, PP2A activation by ceramide or the sphingosine-like compound FTY720 was sufficient to induce TFE3 nuclear translocation. MS analysis revealed that PP2A dephosphorylates TFEB at several residues, including Ser-109, Ser-114, Ser-122, and Ser-211, thus facilitating TFEB activation. Overall, this work identifies a critical mechanism that activates TFEB and TFE3 without turning off mTORC1 activity. We propose that this mechanism may enable some cell types such as immune or cancer cells that require simultaneous TFEB/TFE3 and mTORC1 signaling to survive and achieve robust cell growth in stressful environments.
    Subjects
    Animals; Arsenites - pharmacology; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors - genetics; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors - metabolism; Biochemistry; Cell Biology; Cells, Cultured; Humans; Mice; Molecular Biology; mTORC1; nuclear translocation; okadaic acid; Oxidative Stress; phosphatase; Phosphorylation; protein phosphatase 2 (PP2A); Protein Phosphatase 2 - pharmacology; Signal Transduction; sodium arsenite; Sodium Compounds - pharmacology; TOR Serine-Threonine Kinases - genetics; TOR Serine-Threonine Kinases - metabolism; transcription factor; transcription factor binding to IGHM enhancer 3; transcription factor EB
  • Article

    Introduction of robotic pancreatoduodenectomy following phase 2a IDEAL guidelines

    Authors
    Mise, Yoshihiro; Miyashita, Mamiko; Yoshioka, Ryuji; Kawano, Fumihiro; Takeda, Yoshinori; Ichida, Hirofumi; Saiura, Akio
    Part of
    PloS one, 2024-05, Vol.19 (5), p.e0302848-e0302848
    Abstract
    • Robotic pancreatoduodenectomy (RPD) is a newly introduced procedure, which is still evolving and lacks standardization. An objective assessment is essential to investigate the feasibility of RPD. The current study aimed to assess our initial ten cases of RPD based on IDEAL (Idea, Development, Exploration, Assessment, and Long-term study) guidelines. This was a prospective phase 2a study following the IDEAL framework. Ten consecutive cases of RPD performed by two surgeons with expertise in open procedures at a single center were assigned to the study. With objective evaluation, each case was classified into four grades according to the achievements of the procedures. Errors observed in the previous case were used to inform the procedure in the next case. The surgical outcomes of the ten cases were reviewed. The median total operation time was 634 min (interquartile range [IQR], 594-668) with a median resection time of 363 min (IQR, 323-428) and reconstruction time of 123 min (IQR, 107-131). The achievement of the whole procedure was graded as A, "successful", in two patients. In two patients, reconstruction was performed with a mini-laparotomy due to extensive pneumoperitoneum, probably caused by insertion of a liver retractor from the xyphoid. Major postoperative complications occurred in two patients. One patient, in whom the jejunal limb was elevated through the Treitz ligament, had a bowel obstruction and needed to undergo re-laparotomy. RPD is feasible when performed by surgeons experienced in open procedures. Specific considerations are needed to safely introduce RPD.
    Subjects
    Adult; Aged; Biology and Life Sciences; Female; Gastrointestinal diseases; Humans; Liver; Male; Medical colleges; Medical research; Medicine and Health Sciences; Medicine, Experimental; Methods; Middle Aged; Operative Time; Pancreatic Neoplasms - pathology; Pancreatic Neoplasms - surgery; Pancreaticoduodenectomy; Pancreaticoduodenectomy - adverse effects; Pancreaticoduodenectomy - methods; People and Places; Practice guidelines (Medicine); Prospective Studies; Robotic surgery; Robotic Surgical Procedures - methods; Surgeons; Technology application; Treatment Outcome
  • Article

    IFN- alpha 2a or IFN- beta 1a in combination with ribavirin to treat Middle East respiratory syndrome coronavirus pneumonia: a retrospective study

    Authors
    Shalhoub, Sarah; Farahat, Fayssal; Al-Jiffri, Abdullah; Simhairi, Raed; Shamma, Omar; Siddiqi, Nauman; Mushtaq, Adnan
    Part of
    Journal of antimicrobial chemotherapy, 2015-07, Vol.70 (7), p.2129-2132
    Abstract
    • Objectives Middle East respiratory syndrome coronavirus (MERS-CoV) is associated with significant mortality. We examined the utility of plasma MERS-CoV PCR as a prognostic indicator and compared the efficacies of IFN- alpha 2a and IFN- beta 1a when combined with ribavirin in reducing MERS-CoV-related mortality rates. Methods We retrospectively analysed 32 patients with confirmed MERS-CoV infection, admitted between April 2014 and June 2014, by positive respiratory sample RT-PCR. Plasma MERS-CoV RT-PCR was performed at the time of diagnosis for 19 patients. Results The overall mortality rate was 69% (22/32). Ninety percent (9/10) of patients with positive plasma MERS-CoV PCR died compared with 44% (4/9) of those with negative plasma MERS-CoV PCR. Mortality rate in patients who received IFN- alpha 2a was 85% (11/13) compared with 64% (7/11) in those who received IFN- beta 1a (P=0.24). The mortality rate in patients with renal failure (14), including 8 on haemodialysis, was 100%. Age >50 years and diabetes mellitus were found to be significantly associated with mortality (OR=26.1; 95% CI 3.58-190.76; P=0.001 and OR=15.74; 95% CI 2.46-100.67; P=0.004, respectively). The median duration of viral shedding in patients who recovered was 11 days (range 6-38 days). Absence of fever was noted in 5/32 patients. Conclusions Plasma MERS-CoV RT-PCR may serve as an effective tool to predict MERS-CoV-associated mortality. Older age and comorbid conditions may have contributed to the lack of efficacy of IFN- alpha 2a or IFN- beta 1a with ribavirin in treating MERS-CoV. Absence of fever should not exclude MERS-CoV.
    Subjects
    Coronavirus
  • Article

    B''/PR72 subunit mediates Ca²⁺-dependent dephosphorylation of DARPP-32 by protein phosphatase 2A

    Authors
    Ahn, Jung-Hyuck; Sung, Jee Young; McAvoy, Thomas; Nishi, Akinori; Janssens, Veerle; Goris, Jozef; Greengard, Paul; Nairn, Angus C
    Part of
    Proceedings of the National Academy of Sciences, 2007-06, Vol.104 (23), p.9876-9881
    Abstract
    • In dopaminoceptive neurons, dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32) plays a central role in integrating the effects of dopamine and other neurotransmitters. Phosphorylation of DARPP-32 at Thr-34 by protein kinase A results in inhibition of protein phosphatase 1 (PP1), and phosphorylation at Thr-75 by Cdk5 (cyclin-dependent kinase 5) results in inhibition of protein kinase A. Dephosphorylation at Thr-34 involves primarily the Ca²⁺-dependent protein phosphatase, PP2B (calcineurin), whereas dephosphorylation of Thr-75 involves primarily PP2A, the latter being subject to control by both cAMP- and Ca²⁺-dependent regulatory mechanisms. In the present study, we have investigated the mechanism of Ca²⁺-dependent regulation of Thr-75 by PP2A. We show that the PR72 (or B'' or PPP2R3A) regulatory subunit of PP2A is highly expressed in striatum. Through the use of overexpression and down-regulation by using RNAi, we show that PP2A, in a heterotrimeric complex with the PR72 subunit, mediates Ca²⁺-dependent dephosphorylation at Thr-75 of DARPP-32. The PR72 subunit contains two Ca²⁺ binding sites formed by E and F helices (EF-hands 1 and 2), and we show that the former is necessary for the ability of PP2A activity to be regulated by Ca²⁺, both in vitro and in vivo. Our studies also indicate that the PR72-containing form of PP2A is necessary for the ability of glutamate acting at α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid and NMDA receptors to regulate Thr-75 dephosphorylation. These studies further our understanding of the complex signal transduction pathways that regulate DARPP-32. In addition, our studies reveal an alternative intracellular mechanism whereby Ca²⁺ can activate serine/threonine phosphatase activity.
    Subjects
    Antibodies; Biological Sciences; Calcium - metabolism; Cell Line; Dopamine and cAMP-Regulated Phosphoprotein 32 - metabolism; Down regulation; HEK293 cells; Humans; Immunoblotting; Immunoprecipitation; Multidisciplinary; N methyl D aspartate receptors; Neurons; Neurons - metabolism; Oligonucleotides; Phosphatases; Phosphorylation; Physiological regulation; Protein Phosphatase 2 - metabolism; Protein Subunits - metabolism; RNA Interference; Substrate specificity
  • Article

    Cytoplasmic Retention of Protein Phosphatase 2A Inhibitor 2 (I2PP2A) Induces Alzheimer-like Abnormal Hyperphosphorylation of Tau

    Authors
    Arif, Mohammad; Wei, Jianshe; Zhang, Qi; Liu, Fei; Basurto-Islas, Gustavo; Grundke-Iqbal, Inge; Iqbal, Khalid
    Part of
    Journal of Biological Chemistry, 2014-10, Vol.289 (40), p.27677-27691
    Abstract
    • Abnormal hyperphosphorylation of Tau leads to the formation of neurofibrillary tangles, a hallmark of Alzheimer disease (AD), and related tauopathies. The phosphorylation of Tau is regulated by protein phosphatase 2A (PP2A), which in turn is modulated by endogenous inhibitor 2 (I2PP2A). In AD brain, I2PP2A is translocated from neuronal nucleus to cytoplasm, where it inhibits PP2A activity and promotes abnormal phosphorylation of Tau. Here we describe the identification of a potential nuclear localization signal (NLS) in the C-terminal region of I2PP2A containing a conserved basic motif, 179RKR181, which is sufficient for directing its nuclear localization. The current study further presents an inducible cell model (Tet-Off system) of AD-type abnormal hyperphosphorylation of Tau by expressing I2PP2A in which the NLS was inactivated by 179RKR181 → AAA along with 168KR169 → AA mutations. In this model, the mutant NLS (mNLS)-I2PP2A (I2PP2AAA-AAA) was retained in the cell cytoplasm, where it physically interacted with PP2A and inhibited its activity. Inhibition of PP2A was associated with the abnormal hyperphosphorylation of Tau, which resulted in microtubule network instability and neurite outgrowth impairment. Expression of mNLS-I2PP2A activated CAMKII and GSK-3β, which are Tau kinases regulated by PP2A. The immunoprecipitation experiments showed the direct interaction of I2PP2A with PP2A and GSK-3β but not with CAMKII. Thus, the cell model provides insights into the nature of the potential NLS and the mechanistic relationship between I2PP2A-induced inhibition of PP2A and hyperphosphorylation of Tau that can be utilized to develop drugs preventing Tau pathology.
    Subjects
    Alzheimer Disease - genetics; Alzheimer Disease - metabolism; Biochemistry; Brain - metabolism; Cell Biology; Cell Nucleus - metabolism; Cytoplasm - genetics; Cytoplasm - metabolism; DNA-Binding Proteins; Glycogen Synthase Kinase 3 - genetics; Glycogen Synthase Kinase 3 - metabolism; Glycogen Synthase Kinase 3 beta; Histone Chaperones - genetics; Histone Chaperones - metabolism; Humans; Molecular Biology; Neurobiology; Nuclear Localization Signals; Phosphorylation; Protein Phosphatase 2 - genetics; Protein Phosphatase 2 - metabolism; tau Proteins - genetics; tau Proteins - metabolism; Transcription Factors - genetics; Transcription Factors - metabolism
  • Article

    How allosteric control of Staphylococcus aureus penicillin binding protein 2a enables methicillin resistance and physiological function

    Authors
    Otero, Lisandro H.; Rojas-Altuve, Alzoray; Llarrull, Leticia I.; Carrasco-López, Cesar; Kumarasiri, Malika; Lastochkin, Elena; Fishovitz, Jennifer; Dawley, Matthew; Hesek, Dusan; Lee, Mijoon; Johnson, Jarrod W.; Fisher, Jed F.; Chang, Mayland; Mobashery, Shahriar; Hermoso, Juan A.
    Part of
    Proceedings of the National Academy of Sciences - PNAS, 2013-10, Vol.110 (42), p.16808-16813
    Abstract
    • The expression of penicillin binding protein 2a (PBP2a) is the basis for the broad clinical resistance to the β-lactam antibiotics by methicillin-resistant Staphylococcus aureus (MRSA). The high-molecular mass penicillin binding proteins of bacteria catalyze in separate domains the transglycosylase and transpeptidase activities required for the biosynthesis of the peptidoglycan polymer that comprises the bacterial cell wall. In bacteria susceptible to β-lactam antibiotics, the transpeptidase activity of their penicillin binding proteins (PBPs) is lost as a result of irreversible acylation of an active site serine by the β-lactam antibiotics. In contrast, the PBP2a of MRSA is resistant to β-lactam acylation and successfully catalyzes the dd -transpeptidation reaction necessary to complete the cell wall. The inability to contain MRSA infection with β-lactam antibiotics is a continuing public health concern. We report herein the identification of an allosteric binding domain—a remarkable 60 Å distant from the dd -transpeptidase active site—discovered by crystallographic analysis of a soluble construct of PBP2a. When this allosteric site is occupied, a multiresidue conformational change culminates in the opening of the active site to permit substrate entry. This same crystallographic analysis also reveals the identity of three allosteric ligands: muramic acid (a saccharide component of the peptidoglycan), the cell wall peptidoglycan, and ceftaroline, a recently approved anti-MRSA β-lactam antibiotic. The ability of an anti-MRSA β-lactam antibiotic to stimulate allosteric opening of the active site, thus predisposing PBP2a to inactivation by a second β-lactam molecule, opens an unprecedented realm for β-lactam antibiotic structure-based design.
    Subjects
    Active sites; Acylation; Acylation - physiology; Allosteric Regulation - physiology; Allosteric site; Antibiotics; Bacterial proteins; Binding sites; Biological Sciences; Biosynthesis; Catalytic Domain; Ceftaroline; Cell walls; Cephalosporins - chemistry; Cephalosporins - metabolism; Crystal structure; Crystallography, X-Ray; Gene expression; Ligands; Methicillin Resistance - physiology; Methicillin-Resistant Staphylococcus aureus - enzymology; Methicillin-Resistant Staphylococcus aureus - genetics; Muramic Acids - chemistry; Muramic Acids - metabolism; Penicillin; Penicillin-Binding Proteins - chemistry; Penicillin-Binding Proteins - genetics; Penicillin-Binding Proteins - metabolism; Peptidoglycan - chemistry; Peptidoglycan - metabolism; Physiology; Salts; Staphylococcus aureus; Staphylococcus infections; Substrate Specificity - physiology
  • Article

    Functional Selectivity in Serotonin Receptor 2A (5-HT2A) Endocytosis, Recycling, and Phosphorylation

    Authors
    Raote, Ishier; Bhattacharyya, Samarjit; Panicker, Mitradas M
    Part of
    Molecular Pharmacology, 2013-01, Vol.83 (1), p.42-50
    Abstract
    • G protein-coupled receptor (GPCR) signaling is modulated by endocytosis and endosomal sorting of receptors between degradation and recycling. Differential regulation of these processes by endogenous ligands and synthetic drugs is a poorly understood area of GPCR signaling. Here, we describe remarkable diversity in the regulation of trafficking of GPCR induced by multiple ligands. We show that the serotonin receptor 2A (5-HT(2A)), a prototypical GPCR in the study of functional selectivity at a signaling receptor, is functionally selective in endocytosis and recycling in response to five ligands tested: endogenous agonists serotonin (5-HT) and dopamine (DA), synthetic agonist 1-(2,5-dimethoxy-4-iodophenyl)-aminopropane (DOI), antagonist ketanserin, and inverse agonist and antipsychotic drug clozapine. Only four ligands (5-HT, DA, DOI, and clozapine) bring about receptor endocytosis. As we have earlier described with 5-HT and DA, there is ligand-specific requirement for protein kinase C (PKC) in endocytosis. We now show 5-HT(2A) phosphorylation by PKC is necessary for 5-HT-mediated and DOI-mediated receptor endocytosis, but DA-mediated and clozapine-mediated internalization is not affected if PKC is inhibited. Internalized receptors are recycled to the cell surface, but there is variability in the time course of recycling. 5-HT- and DA-internalized receptors are recycled in 2.5 hours while agonist DOI and antagonist clozapine bring about recycling in 7.5 hours. Recycling in response to those ligands that require PKC activation to effect receptor endocytosis is dependent on receptor dephosphorylation by protein phosphatase 2A (PP2A). Thus, internalization and phosphorylation/dephosphorylation cycles may play a significant role in the regulation of 5-HT(2A) by functionally and therapeutically important ligands.
    Subjects
    Amphetamines - pharmacology; Animals; Clozapine - pharmacology; Dopamine - pharmacology; Drug Partial Agonism; Endocytosis; Enzyme Activation; HEK293 Cells; Humans; Ketanserin - pharmacology; Ligands; Molecular Medicine; Pharmacology; Phosphorylation; Protein Kinase C - metabolism; Protein Phosphatase 2 - metabolism; Protein Transport; Rats; Receptor, Serotonin, 5-HT2A - metabolism; Serotonin - pharmacology; Serotonin 5-HT2 Receptor Agonists - pharmacology; Serotonin 5-HT2 Receptor Antagonists - pharmacology; Signal Transduction
  • Article

    A new paradigm for regulation of protein phosphatase 2A function via Src and Fyn kinase–mediated tyrosine phosphorylation

    Authors
    Sontag, Jean-Marie; Schuhmacher, Diana; Taleski, Goce; Jordan, Anthony; Khan, Sarah; Hoffman, Alexander; Gomez, Rey J.; Mazalouskas, Matthew D.; Hanks, Steven K.; Spiller, Benjamin W.; Sontag, Estelle; Wadzinski, Brian E.
    Part of
    The Journal of biological chemistry, 2022-08, Vol.298 (8), p.102248-102248, Article 102248
    Abstract
    • Protein phosphatase 2A (PP2A) is a major phospho-Ser/Thr phosphatase and a key regulator of cellular signal transduction pathways. While PP2A dysfunction has been linked to human cancer and neurodegenerative disorders such as Alzheimer’s disease (AD), PP2A regulation remains relatively poorly understood. It has been reported that the PP2A catalytic subunit (PP2Ac) is inactivated by a single phosphorylation at the Tyr307 residue by tyrosine kinases such as v-Src. However, multiple mass spectrometry studies have revealed the existence of other putative PP2Ac phosphorylation sites in response to activation of Src and Fyn, two major Src family kinases (SFKs). Here, using PP2Ac phosphomutants and novel phosphosite-specific PP2Ac antibodies, we show that cellular pools of PP2Ac are instead phosphorylated on both Tyr127 and Tyr284 upon Src activation, and on Tyr284 following Fyn activation. We found these phosphorylation events enhanced the interaction of PP2Ac with SFKs. In addition, we reveal SFK-mediated phosphorylation of PP2Ac at Y284 promotes dissociation of the regulatory Bα subunit, altering PP2A substrate specificity; the phosphodeficient Y127/284F and Y284F PP2Ac mutants prevented SFK-mediated phosphorylation of Tau at the CP13 (pSer202) epitope, a pathological hallmark of AD, and SFK-dependent activation of ERK, a major growth regulatory kinase upregulated in many cancers. Our findings demonstrate a novel PP2A regulatory mechanism that challenges the existing dogma on the inhibition of PP2A catalytic activity by Tyr307 phosphorylation. We propose dysregulation of SFK signaling in cancer and AD can lead to alterations in PP2A phosphorylation and subsequent deregulation of key PP2A substrates, including ERK and Tau.
    Subjects
    ERK; Fyn; PP2A; protein phosphatase 2A; Src; Tau; tyrosine phosphorylation
  • Article

    γ-H2AX Dephosphorylation by Protein Phosphatase 2A Facilitates DNA Double-Strand Break Repair

    Authors
    Chowdhury, Dipanjan; Keogh, Michael-Christopher; Ishii, Haruhiko; Peterson, Craig L.; Buratowski, Stephen; Lieberman, Judy
    Part of
    Molecular cell, 2005-12, Vol.20 (5), p.801-809
    Abstract
    • Phosphorylated histone H2AX (γ-H2AX) forms foci over large chromatin domains surrounding double-stranded DNA breaks (DSB). These foci recruit DSB repair proteins and dissolve during or after repair is completed. How γ-H2AX is removed from chromatin remains unknown. Here, we show that protein phosphatase 2A (PP2A) is involved in removing γ-H2AX foci. The PP2A catalytic subunit [PP2A(C)] and γ-H2AX coimmunoprecipitate and colocalize in DNA damage foci and PP2A dephosphorylates γ-H2AX in vitro. The recruitment of PP2A(C) to DNA damage foci is H2AX dependent. When PP2A(C) is inhibited or silenced by RNA interference, γ-H2AX foci persist, DNA repair is inefficient, and cells are hypersensitive to DNA damage. The effect of PP2A on γ-H2AX levels is independent of ATM, ATR, or DNA-PK activity.
    Subjects
    Animals; Ataxia Telangiectasia Mutated Proteins; Cell Cycle Proteins - metabolism; Cell Line; DNA Damage; DNA Repair; DNA-Activated Protein Kinase - metabolism; DNA-Binding Proteins - metabolism; Fibroblasts; HeLa Cells; Histones - deficiency; Histones - metabolism; Humans; In Vitro Techniques; Mice; Phosphoprotein Phosphatases - metabolism; Phosphorylation; Protein Phosphatase 2; Protein-Serine-Threonine Kinases - metabolism; Tumor Suppressor Proteins - metabolism
  • Article

    Agonist-directed signaling of the serotonin 2A receptor depends on β-arrestin-2 interactions in vivo

    Authors
    Schmid, Cullen L; Raehal, Kirsten M; Bohn, Laura M
    Part of
    Proceedings of the National Academy of Sciences - PNAS, 2008-01, Vol.105 (3), p.1079-1084
    Abstract
    • Visual and auditory hallucinations accompany certain neuropsychiatric disorders, such as schizophrenia, and they also can be induced by the use or abuse of certain drugs. The heptahelical serotonin 2A receptors (5-HT2ARs) are molecular targets for drug-induced hallucinations. However, the cellular mechanisms by which the 5-HT2AR mediates these effects are not well understood. Drugs acting at the 5-HT2AR can trigger diverse signaling pathways that may be directed by the chemical properties of the drug. β-arrestins are intracellular proteins that bind to heptahelical receptors and represent a point where such divergences in ligand-directed functional signaling could occur. Here we compare the endogenous agonist, serotonin, to a synthetic 5-HT2AR hallucinogenic agonist, 2,5-dimethoxy-4-iodoamphetamine (DOI), in mice lacking β-arrestin-2, as well as in cells lacking β-arrestins. In mice, we find that serotonin induces a head twitch response by a β-arrestin-2-dependent mechanism. However, DOI invokes the behavior independent of β-arrestin-2. The two structurally distinct agonists elicit different signal transduction and trafficking patterns upon activation of 5-HT2AR, which hinge on the presence of β-arrestins. Our study suggests that the 5-HT2AR-β-arrestin interaction may be particularly important in receptor function in response to endogenous serotonin levels, which could have major implications in drug development for treating neuropsychiatric disorders such as depression and schizophrenia.
    Subjects
    Agonists; Animals; Arrestins - deficiency; Arrestins - genetics; Arrestins - metabolism; beta-Arrestin 2; beta-Arrestins; Biological Sciences; Cell Line; Genotypes; Internalization; Mice; Mice, Knockout; Mitogen-Activated Protein Kinase 1 - metabolism; Mitogen-Activated Protein Kinase 3 - metabolism; Neurons; Phosphorylation; Phosphorylation - drug effects; Protein Binding; Receptor, Serotonin, 5-HT2A - metabolism; Receptors; Serotonin 5-HT2 Receptor Agonists; Serotonin agents; Serotonin receptors; Signal Transduction - drug effects; Vehicles
  • Article

    Synthesis and biological investigation of protein phosphatase 2A‐activating compounds with dimeric tail as non‐small cell lung cancer cell death agents

    Authors
    Kim, Su Bin; Lee, Taeho; Oh, Yoon Sin; Park, Eun‐Young; Baek, Dong Jae
    Part of
    Bulletin of the Korean Chemical Society, 2023, 44(4), , pp.293-297
    Abstract
    • FTY720 exerts an anticancer effect through the activation of protein phosphatase 2A (PP2A), which acts as a tumor suppressor, and inhibits SK1 activity. However, FTY720 taken into the body is phosphorylated by endogenous SK2. The structure of FTY720 is well‐studied as a basis for developing SK inhibitors and PP2A activators. We synthesized analogs of PF‐543 with a dimeric tail structure and reported the efficacy of SK1 inhibition. The compounds with this structure had improved anticancer activity and stability compared with PF‐543. To confirm whether the dimeric tail structure could be applied as a PP2A‐active material, we synthesized four types of compounds with a dimeric tail structure and confirmed the anticancer activity and PP2A activation in A549 non‐small cell lung cancer cells. Compounds 2 and 4 were more cytotoxic to A549 cells than RB005, and induced similar levels of cytotoxic and PP2A activation as FTY720. FTY720 analogs were synthesized by introducing a dimeric aliphatic tail into the FTY720 backbone, and their biological activities were investigated. Compound 4 showed more cytotoxicity against lung cancer cell line A549 than RB005 (SK1 inhibitor), and showed a similar level of cytotoxicity to FTY720. Compound 4 induced PP2A activation, which is one of the anticancer mechanisms of FTY720, and showed PP2A docking results similar to those of FTY720.
    Subjects
    FTY720; non‐small cell lung cancer; PF‐543; protein phosphatase 2A; sphingosine kinase; 화학
  • Article

    Biguanide metformin acts on tau phosphorylation via mTOR/protein phosphatase 2A (PP2A) signaling

    Authors
    Kickstein, Eva; Krauss, Sybille; Thornhill, Paul; Rutschow, Désirée; Zeller, Raphael; Sharkey, John; Williamson, Ritchie; Fuchs, Melanie; Köhler, Andrea; Glossmann, Hartmut; Schneider, Rainer; Sutherland, Calum; Schweiger, Susann; Südhof, Thomas C.
    Part of
    Proceedings of the National Academy of Sciences, 2010-12, Vol.107 (50), p.21830-21835
    Abstract
    • Hyperphosphorylated tau plays an important role in the formation of neurofibrillary tangles in brains of patients with Alzheimer's disease (AD) and related tauopathies and is a crucial factor in the pathogenesis of these disorders. Though diverse kinases have been implicated in tau phosphorylation, protein phosphatase 2A (PP2A) seems to be the major tau phosphatase. Using murine primary neurons from wild-type and human tau transgenic mice, we show that the antidiabetic drug metformin induces PP2A activity and reduces tau phosphorylation at PP2A-dependent epitopes in vitro and in vivo. This tau dephosphorylating potency can be blocked entirely by the PP2A inhibitors okadaic acid and fostriecin, confirming that PP2A is an important mediator of the observed effects. Surprisingly, metformin effects on PP2A activity and tau phosphorylation seem to be independent of AMPK activation, because in our experiments (i) metformin induces PP2A activity before and at lower levels than AMPK activity and (ii) the AMPK activator AICAR does not influence the phosphorylation of tau at the sites analyzed. Affinity chromatography and immunoprecipitation experiments together with PP2A activity assays indicate that metformin interferes with the association of the catalytic subunit of PP2A (PP2Ac) to the so-called MID1-α4 protein complex, which regulates the degradation of PP2Ac and thereby influences PP2A activity. In summary, our data suggest a potential beneficial role of biguanides such as metformin in the prophylaxis and/or therapy of AD.
    Subjects
    Actins; Adenylate Kinase - metabolism; Alzheimer Disease - metabolism; Alzheimer Disease - pathology; Alzheimer Disease - physiopathology; Alzheimer's disease; Alzheimers disease; Animals; Antibodies; Biological Sciences; Brain; Cells, Cultured; Enzyme Inhibitors - pharmacology; Epitopes; HeLa Cells; Humans; Hypoglycemic Agents - pharmacology; Insulin; Kinases; Mechanistic Target of Rapamycin Complex 1; Metformin - pharmacology; Mice; Mice, Transgenic; Multidisciplinary; Multiprotein Complexes; Neurofibrillary Tangles - metabolism; Neurofibrillary Tangles - pathology; Neurons; Neurons - cytology; Neurons - metabolism; Okadaic Acid - pharmacology; Phosphatases; Phosphorylation; Physiological regulation; Protein Phosphatase 2 - genetics; Protein Phosphatase 2 - metabolism; Proteins; Proteins - metabolism; Signal transduction; Signal Transduction - drug effects; tau Proteins - genetics; tau Proteins - metabolism; TOR Serine-Threonine Kinases - genetics; TOR Serine-Threonine Kinases - metabolism

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