DOCSLIB.ORG

Mouse Ppp2r2a Conditional Knockout Project (CRISPR/Cas9)

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Mouse Ppp2r2a Conditional Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Ppp2r2a Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Ppp2r2a conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Ppp2r2a gene (NCBI Reference Sequence: NM_028032 ; Ensembl: ENSMUSG00000022052 ) is located on Mouse chromosome 14. 10 exons are identified, with the ATG start codon in exon 1 and the TAG stop codon in exon 10 (Transcript: ENSMUST00000089230). Exon 4 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Ppp2r2a gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-62C15 as template. Cas9, gRNA and targeting vector will be co-injected into fertilized eggs for cKO Mouse production. The pups will be genotyped by PCR followed by sequencing analysis. Note: Exon 4 starts from about 13.5% of the coding region. The knockout of Exon 4 will result in frameshift of the gene. The size of intron 3 for 5'-loxP site insertion: 9844 bp, and the size of intron 4 for 3'-loxP site insertion: 4224 bp. The size of effective cKO region: ~666 bp. The cKO region does not have any other known gene. Page 1 of 7 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele gRNA region 5' gRNA region 3' 1 4 10 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Ppp2r2a Homology arm cKO region loxP site Page 2 of 7 https://www.alphaknockout.com Overview of the Dot Plot Window size: 10 bp Forward Reverse Complement Sequence 12 Note: The sequence of homologous arms and cKO region is aligned with itself to determine if there are tandem repeats. No significant tandem repeat is found in the dot plot matrix. So this region is suitable for PCR screening or sequencing analysis. Overview of the GC Content Distribution Window size: 300 bp Sequence 12 Summary: Full Length(7166bp) | A(26.46% 1896) | C(18.11% 1298) | T(34.13% 2446) | G(21.3% 1526) Note: The sequence of homologous arms and cKO region is analyzed to determine the GC content. No significant high GC-content region is found. So this region is suitable for PCR screening or sequencing analysis. Page 3 of 7 https://www.alphaknockout.com BLAT Search Results (up) QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ----------------------------------------------------------------------------------------------- browser details YourSeq 3000 1 3000 3000 100.0% chr14 - 67029281 67032280 3000 browser details YourSeq 139 1230 1396 3000 94.5% chr4 - 106844723 106845094 372 browser details YourSeq 139 1217 1399 3000 89.8% chr16 - 95518244 95518460 217 browser details YourSeq 134 1227 1380 3000 94.8% chr12 - 111472212 111472397 186 browser details YourSeq 133 1104 1380 3000 92.4% chr2 - 72968172 72968602 431 browser details YourSeq 132 1225 1392 3000 91.9% chr9 - 22466604 22466802 199 browser details YourSeq 126 1228 1377 3000 93.9% chr10 - 12885129 12885307 179 browser details YourSeq 125 1222 1380 3000 91.4% chr12 + 22018235 22018418 184 browser details YourSeq 125 1227 1413 3000 91.9% chr10 + 111256920 111257106 187 browser details YourSeq 124 1225 1399 3000 88.8% chr13 - 119441680 119441932 253 browser details YourSeq 123 1230 1380 3000 94.3% chr2 - 180649581 180649752 172 browser details YourSeq 123 1228 1380 3000 94.9% chr11 - 90688925 90689106 182 browser details YourSeq 122 1218 1380 3000 90.7% chr9 - 58235523 58235706 184 browser details YourSeq 122 1217 1380 3000 90.7% chr5 + 79970961 79971155 195 browser details YourSeq 121 1217 1380 3000 89.1% chr13 - 115883347 115883548 202 browser details YourSeq 121 1218 1380 3000 91.1% chr4 + 143236345 143236521 177 browser details YourSeq 118 1227 1360 3000 94.1% chr13 - 24403951 24404084 134 browser details YourSeq 117 1227 1380 3000 93.4% chr15 + 83096695 83096874 180 browser details YourSeq 116 1242 1380 3000 92.1% chr9 - 101917794 101917951 158 browser details YourSeq 116 1227 1358 3000 94.7% chr17 - 74459322 74459488 167 Note: The 3000 bp section upstream of Exon 4 is BLAT searched against the genome. No significant similarity is found. BLAT Search Results (down) QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN -------------------------------------------------------------------------------------------------------------- browser details YourSeq 3000 1 3000 3000 100.0% chr14 - 67025615 67028614 3000 browser details YourSeq 176 1604 1795 3000 96.4% chr14 - 73229455 73229659 205 browser details YourSeq 175 1583 1776 3000 96.4% chr8 + 111647670 111647874 205 browser details YourSeq 173 1589 1783 3000 94.9% chr11 + 95295621 95295830 210 browser details YourSeq 172 1590 1790 3000 93.9% chr16 + 13803452 13803658 207 browser details YourSeq 171 1590 1793 3000 93.7% chr2 + 79721943 79722144 202 browser details YourSeq 169 1594 1776 3000 96.2% chr5 - 137479017 137479199 183 browser details YourSeq 168 1594 1775 3000 96.2% chr19 - 7007499 7007680 182 browser details YourSeq 168 1596 1775 3000 97.3% chr16 + 38382898 38383503 606 browser details YourSeq 168 1590 1776 3000 92.9% chr10 + 115895229 115895409 181 browser details YourSeq 167 1590 1775 3000 95.7% chr9 - 72126619 72126990 372 browser details YourSeq 167 1594 1784 3000 93.8% chr11 + 88858046 88858236 191 browser details YourSeq 166 1602 1789 3000 94.2% chr9 + 100632841 100633028 188 browser details YourSeq 166 1590 1775 3000 95.2% chr7 + 28625928 28626127 200 browser details YourSeq 166 1597 1775 3000 96.7% chr12 + 70974154 70974333 180 browser details YourSeq 165 1604 1783 3000 96.7% chr4 + 40838574 40838754 181 browser details YourSeq 164 1589 1775 3000 91.7% chr2 + 157226713 157226893 181 browser details YourSeq 164 1583 1774 3000 93.7% chr10 + 37700003 37700198 196 browser details YourSeq 163 1594 1783 3000 93.7% chrX - 150227131 150227320 190 browser details YourSeq 163 1589 1775 3000 91.9% chr8 + 22412875 22413058 184 Note: The 3000 bp section downstream of Exon 4 is BLAT searched against the genome. No significant similarity is found. Page 4 of 7 https://www.alphaknockout.com Gene and protein information: Ppp2r2a protein phosphatase 2, regulatory subunit B, alpha [ Mus musculus (house mouse) ] Gene ID: 71978, updated on 24-Oct-2019 Gene summary Official Symbol Ppp2r2a provided by MGI Official Full Name protein phosphatase 2, regulatory subunit B, alpha provided by MGI Primary source MGI:MGI:1919228 See related Ensembl:ENSMUSG00000022052 Gene type protein coding RefSeq status VALIDATED Organism Mus musculus Lineage Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Myomorpha; Muroidea; Muridae; Murinae; Mus; Mus Also known as mKIAA1541; 2410004D02Rik Expression Ubiquitous expression in adrenal adult (RPKM 26.6), CNS E11.5 (RPKM 11.2) and 27 other tissues See more Orthologs human all Genomic context Location: 14; 14 D1 See Ppp2r2a in Genome Data Viewer Exon count: 11 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 14 NC_000080.6 (67014056..67072471, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 14 NC_000080.5 (67634508..67691266, complement) Chromosome 14 - NC_000080.6 Page 5 of 7 https://www.alphaknockout.com Transcript information: This gene has 5 transcripts Gene: Ppp2r2a ENSMUSG00000022052 Description protein phosphatase 2, regulatory subunit B, alpha [Source:MGI Symbol;Acc:MGI:1919228] Gene Synonyms 2410004D02Rik Location Chromosome 14: 67,014,056-67,072,444 reverse strand. GRCm38:CM001007.2 About this gene This gene has 5 transcripts (splice variants), 146 orthologues, 3 paralogues, is a member of 1 Ensembl protein family and is associated with 7 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Ppp2r2a- ENSMUST00000089230.6 4010 447aa ENSMUSP00000086640.5 Protein coding CCDS56967 Q3TT94 TSL:1 201 Q6P1F6 GENCODE basic APPRIS P1 Ppp2r2a- ENSMUST00000225380.1 1988 367aa ENSMUSP00000153191.1 Protein coding - Q9CWU3 GENCODE 204 basic Ppp2r2a- ENSMUST00000225251.1 2192 50aa ENSMUSP00000153452.1 Nonsense mediated - A0A286YDJ9 - 203 decay Ppp2r2a- ENSMUST00000223630.1 1845 No - Retained intron - - - 202 protein Ppp2r2a- ENSMUST00000225469.1 770 No - lncRNA - - - 205 protein 78.39 kb Forward strand Genes Gm27647-201 >miRNA (Comprehensive set... Contigs AC165148.2 > < CT030233.6 Genes (Comprehensive set... < Bnip3l-201protein codin<g Ppp2r2a-204protein coding < Bnip3l-202protein coding< Ppp2r2a-203nonsense mediated decay < Ppp2r2a-201protein coding < Ppp2r2a-202retained intron < Ppp2r2a-205lncRNA Regulatory Build Reverse strand 78.39 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Gene Legend Protein Coding Ensembl protein coding merged Ensembl/Havana Non-Protein Coding processed transcript RNA gene Page 6 of 7 https://www.alphaknockout.com Transcript: ENSMUST00000089230 < Ppp2r2a-201protein coding Reverse strand 58.39 kb ENSMUSP00000086... Low complexity (Seg) Superfamily WD40-repeat-containing domain superfamily SMART WD40 repeat Prints Protein phosphatase 2A regulatory subunit PR55 PROSITE patterns Protein phosphatase 2A regulatory subunit PR55, conserved site Protein phosphatase 2A regulatory subunit PR55, conserved site PIRSF Protein phosphatase 2A regulatory subunit PR55 PANTHER Protein phosphatase 2A regulatory subunit PR55 PTHR11871:SF2 Gene3D WD40/YVTN repeat-like-containing domain superfamily All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend splice region variant synonymous variant Scale bar 0 40 80 120 160 200 240 280 320 360 400 447 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 7 of 7.
Load more

Recommended publications
  • Novelty Indicator for Enhanced Prioritization of Predicted Gene Ontology Annotations
    IEEE/ACM TRANSACTIONS ON COMPUTATIONAL BIOLOGY AND BIOINFORMATICS, VOL. X, NO. X, MONTHXXX 20XX 1 Novelty Indicator for Enhanced Prioritization of Predicted Gene Ontology Annotations Davide Chicco, Fernando Palluzzi, and Marco Masseroli Abstract—Biomolecular controlled annotations have become pivotal in computational biology, because they allow scientists to analyze large amounts of biological data to better understand their test results, and to infer new knowledge. Yet, biomolecular annotation databases are incomplete by definition, like our knowledge of biology, and may contain errors and inconsistent information. In this context, machine-learning algorithms able to predict and prioritize new biomolecular annotations are both effective and efficient, especially if compared with the time-consuming trials of biological validation. To limit the possibility that these techniques predict obvious and trivial high-level features, and to help prioritizing their results, we introduce here a new element that can improve the accuracy and relevance of the results of an annotation prediction and prioritization pipeline. We propose a novelty indicator able to state the level of ”newness” (or ”originality”) of the annotations predicted for a specific gene to Gene Ontology terms, and to help prioritizing the most novel and interesting annotations predicted. We performed a thorough biological functional analysis of the prioritized annotations predicted with high accuracy by using this indicator and our previously proposed prediction algorithms. The relevance
    [Show full text]
  • Loss of PPP2R2A Inhibits Homologous Recombination DNA Repair and Predicts
    Author Manuscript Published OnlineFirst on October 18, 2012; DOI: 10.1158/0008-5472.CAN-12-1667 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Loss of PPP2R2A inhibits homologous recombination DNA repair and predicts tumor sensitivity to PARP inhibition Peter Kalev1, Michal Simicek1, Iria Vazquez1, Sebastian Munck1, Liping Chen2, Thomas Soin1, Natasha Danda1, Wen Chen2 and Anna Sablina1,* 1VIB Center for the Biology of Disease; Center for Human Genetics, KULeuven, Leuven 3000 Belgium 2Department of Toxicology, Faculty of Preventive Medicine, Guangdong Provincial Key Laboratory of Food, Nutrition and Health, School of Public Health, Sun Yat-sen University, Guangzhou 510080, China *Corresponding author information: [email protected] Contact information: Anna A. Sablina, Ph.D. CME Department, KULeuven O&N I Herestraat 49, bus 602 Leuven, Belgium 3000 Tel: +3216330790 Fax: +3216330145 Running title: The role of PPP2R2A in DNA repair Keywords: PP2A, ATM, DNA repair, cancer, PARP inhibition Conflict of interests: The authors claim no conflict of interest. - 1 - Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2012 American Association for Cancer Research. Author Manuscript Published OnlineFirst on October 18, 2012; DOI: 10.1158/0008-5472.CAN-12-1667 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Abstract Reversible phosphorylation plays a critical role in DNA repair. Here we report the results of a loss-of-function screen that identifies the PP2A heterotrimeric serine/threonine phosphatases PPP2R2A, PPP2R2D, PPP2R5A and PPP2R3C in double-strand break (DSB) repair. In particular, we found that PPP2R2A-containing complexes directly dephosphorylated ATM at S367, S1893, and S1981 to regulate its retention at DSB sites.
    [Show full text]
  • Patterning and Gastrulation Defects Caused by the Tw18 Lethal Are Due
    © 2017. Published by The Company of Biologists Ltd | Biology Open (2017) 6, 752-764 doi:10.1242/bio.023200 RESEARCH ARTICLE Patterning and gastrulation defects caused by the tw18 lethal are due to loss of Ppp2r1a Lisette Lange1,2,‡, Matthias Marks1,*,‡, Jinhua Liu1, Lars Wittler1, Hermann Bauer1, Sandra Piehl1, Gabriele Bläß1, Bernd Timmermann3 and Bernhard G. Herrmann1,4,§ ABSTRACT t haplotypes, to date only one t lethal, tw5, has been identified and The mouse t haplotype, a variant 20 cM genomic region on characterized at the molecular level (Sugimoto et al., 2012). The t w18 Chromosome 17, harbors 16 embryonic control genes identified by lethal t and related t lethals of the same complementation group 4 9 recessive lethal mutations isolated from wild mouse populations. Due (t , t ) were described about 50 years ago (Bennett and Dunn, 1960; to technical constraints so far only one of these, the tw5 lethal, has Moser and Gluecksohn-Waelsch, 1967). They cause strong been cloned and molecularly characterized. Here we report the gastrulation defects with striking overgrowth of the primitive molecular isolation of the tw18 lethal. Embryos carrying the tw18 lethal streak (PS) and bulging of cells into the amniotic cavity, die from major gastrulation defects commencing with primitive streak commencing on the seventh and prominent on the eighth to ninth formation at E6.5. We have used transcriptome and marker gene day of gestation, followed by embryonic death one day later. In analyses to describe the molecular etiology of the tw18 phenotype. We contrast, normal development requires the ingression of epiblast show that both WNT and Nodal signal transduction are impaired in the cells at the PS, epithelial-to-mesenchymal transition (EMT) and mutant epiblast, causing embryonic patterning defects and failure of migration of single mesodermal cells in between the epiblast and the primitive streak and mesoderm formation.
    [Show full text]
  • Loss of PPP2R2A Inhibits Homologous Recombination DNA Repair and Predicts Tumor Sensitivity to PARP Inhibition
    Published OnlineFirst October 18, 2012; DOI: 10.1158/0008-5472.CAN-12-1667 Cancer Molecular and Cellular Pathobiology Research Loss of PPP2R2A Inhibits Homologous Recombination DNA Repair and Predicts Tumor Sensitivity to PARP Inhibition Peter Kalev1, Michal Simicek1, Iria Vazquez1, Sebastian Munck1, Liping Chen2, Thomas Soin1, Natasha Danda1, Wen Chen2, and Anna Sablina1 Abstract Reversible phosphorylation plays a critical role in DNA repair. Here, we report the results of a loss-of-function screen that identifies the PP2A heterotrimeric serine/threonine phosphatases PPP2R2A, PPP2R2D, PPP2R5A, and PPP2R3C in double-strand break (DSB) repair. In particular, we found that PPP2R2A-containing complexes directly dephosphorylated ATM at S367, S1893, and S1981 to regulate its retention at DSB sites. Increased ATM phosphorylation triggered by PPP2R2A attenuation dramatically upregulated the activity of the downstream effector kinase CHK2, resulting in G1 to S-phase cell-cycle arrest and downregulation of BRCA1 and RAD51. In tumor cells, blocking PPP2R2A thereby impaired the high-fidelity homologous recombination repair pathway and sensitized cells to small-molecule inhibitors of PARP. We found that PPP2R2A was commonly downregulated in non–small cell lung carcinomas, suggesting that PPP2R2A status may serve as a marker to predict therapeutic efficacy to PARP inhibition. In summary, our results deepen understanding of the role of PP2A family phosphatases in DNA repair and suggest PPP2R2A as a marker for PARP inhibitor responses in clinic. Cancer Res; 72(24); 6414–24. Ó2012 AACR. Introduction during the DNA repair process (3, 4), suggesting that phos- Genome stability is essential for the prevention of undue phatases may serve not only as negative regulators but also as cellular death and cancer development.
    [Show full text]
  • Prostaglandin A1 Inhibits the Phosphorylation of Tau Via Activating Protein Phosphotase 2A in a Michael Addition Mechanism at the Site of Cysteine 377
    Prostaglandin A1 inhibits the phosphorylation of tau via activating protein phosphotase 2A in a michael addition mechanism at the site of cysteine 377 Guo-Biao Xu Northeastern University Pei-Pei Guan Northeastern University Pu Wang ( [email protected] ) Northeastern University https://orcid.org/0000-0002-9617-5686 Research Keywords: prostaglandin A1, protein phosphotase 2A scaffold subunit A alpha, michael addition, tau, Alzheimer’s disease Posted Date: August 12th, 2020 DOI: https://doi.org/10.21203/rs.3.rs-56068/v1 License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/26 Abstract Background: Prostaglandin (PG) A1 is a metabolic product of cyclooxygenase 2 (COX-2), which potentially involved in regulating the development and progression of Alzheimer’s disease (AD). As a cyclopentenone (cy) PG, PGA1 is characterized by the presence of a chemically reactive α, β-unsaturated carbonyl. Although PGA1 is potentially involved in regulating multiple biological processes via michael addition, its specic roles in AD remained unclear. Methods: The tauP301S transgenic (Tg) mice were employed as in vivo AD models and neuroblastoma (N) 2a cells as in vitro neuronal models. By intracerebroventricular injected (i.c.v) with PGA1, the binding proteins to PGA1 are analyzed by HPLC-MS-MS. In addition, western blots are used to determine the phosphorylation of tau in PGA1 treated Tg mice in the absence or presence of okadaic acid (OA), an inhibitor of protein phosphotase (PP) 2A. Combining a synthesis of pull down assay, immunoprecipitation, western blots and HPLC-MS-MS, PP2A scaffold subunit A alpha (PPP2R1A) was identied to be activated by directly binding on PGA1 in cysteine 377-dependent manner.
    [Show full text]
  • Review of PP2A Tumor Biology and Antitumor Effects of PP2A Inhibitor LB100 in the Nervous System
    cancers Review Review of PP2A Tumor Biology and Antitumor Effects of PP2A Inhibitor LB100 in the Nervous System Jean-Paul Bryant 1 , Adam Levy 2 , John Heiss 1 and Yeshavanth Kumar Banasavadi-Siddegowda 1,* 1 Surgical Neurology Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892, USA; [email protected] (J.-P.B.); [email protected] (J.H.) 2 Miller School of Medicine, University of Miami, Miami, FL 33136, USA; [email protected] * Correspondence: [email protected]; Tel.: +1-301-451-0970 Simple Summary: Central and peripheral nervous system tumors represent a heterogenous group of neoplasms which often demonstrate resistance to treatment. Given that these tumors are often refractory to conventional therapy, novel pharmaceutical regimens are needed for successfully treating this pathology. One such therapeutic is the serine/threonine phosphatase inhibitor, LB100. LB100 is a water-soluble competitive protein phosphtase inhibitor that has demonstrated antitumor effects in preclinical and clinical trials. In this review, we aim to summarize current evidence demonstrating the efficacy of LB100 as an inhibitor of nervous system tumors. Furthermore, we review the involvement of the well-studied phosphatase, protein phosphatase 2A, in oncogenic cell signaling pathways, neurophysiology, and neurodevelopment. Abstract: Protein phosphatase 2A (PP2A) is a ubiquitous serine/threonine phosphatase implicated in a wide variety of regulatory cellular functions. PP2A is abundant in the mammalian nervous system, and dysregulation of its cellular functions is associated with myriad neurodegenerative Citation: Bryant, J.-P.; Levy, A.; disorders. Additionally, PP2A has oncologic implications, recently garnering attention and emerging Heiss, J.; Banasavadi-Siddegowda, as a therapeutic target because of the antitumor effects of a potent PP2A inhibitor, LB100.
    [Show full text]
  • Ppp2r2a Prostate Cancer Haploinsufficiency Is Associated
    PPP2R2A PROSTATE CANCER HAPLOINSUFFICIENCY IS ASSOCIATED WITH WORSE PROGNOSIS AND A HIGH VULNERABILITY TO B55/PP2A RECONSTITUTION THAT TRIGGERS CENTROSOME DESTABILIZATION AND INHIBITS CELL INVASION A Dissertation Submitted to the Temple University Graduate Board In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY by Ziran Zhao May 2020 Examining Committee Members: Xavier Graña, PhD, Advisory Chair, Fels Institute and Dept. of Biochemistry Scott K. Shore, PhD, Fels Institute and Dept. of Biochemistry Dale S. Haines, PhD, Fels Institute and Dept. of Biochemistry M. Raza Zaidi, PhD, Fels Institute and Dept. of Biochemistry Bojana Gligorijevic, PhD, External Member, College of Engineering ABSTRACT The PPP2R2A gene encodes the B55α regulatory subunit of PP2A. Here we report that PPP2R2A is hemizygously lost in ~42% of prostate adenocarcinomas, correlating with reduced expression, poorer prognosis, and an increased incidence of hemizygous loss (>75%) in metastatic disease. Of note, PPP2R2A homozygous loss is less common (5%) and not increased at later tumor stages. Reduced expression of B55α is also seen in prostate tumor tissue and cell lines. Consistent with the possibility that complete loss of PPP2R2A is detrimental in prostate tumors, PPP2R2A deletion in cells with reduced but present B55α reduces cell proliferation by slowing progression through multiple phases in the cell cycle. Remarkably, B55α-low cells also appear addicted to lower B55α expression, as even moderate increases in B55α expression are toxic. Reconstitution of B55α expression in prostate cancer (PCa) cell lines with low B55α expression reduces proliferation, inhibits transformation and blocks xenograft tumorigenicity. Mechanistically, we show B55α reconstitution reduces phosphorylation of proteins essential for centrosomal maintenance, and induces centrosome collapse and chromosome segregation failure; a first reported link between B55α/PP2A and the vertebrate centrosome.
    [Show full text]
  • Low Expression of PP2A Regulatory Subunit B55&Alpha
    Leukemia (2011) 25, 1711–1717 & 2011 Macmillan Publishers Limited All rights reserved 0887-6924/11 www.nature.com/leu ORIGINAL ARTICLE Low expression of PP2A regulatory subunit B55a is associated with T308 phosphorylation of AKT and shorter complete remission duration in acute myeloid leukemia patients PP Ruvolo1, YH Qui1, KR Coombes2, N Zhang2, VR Ruvolo1, G Borthakur1, M Konopleva1, M Andreeff1 and SM Kornblau1 1Department of Leukemia, University of Texas MD Anderson Cancer Center, Houston, TX, USA and 2Department of Bioinformatics and Computational Biology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA The regulation of protein kinase B (AKT) is a dynamic process by a phosphatidylinositol 3 kinase/phosphatidylinositol-depen- that depends on the balance between phosphorylation by dent kinase 1 cascade that promotes phosphorylation of the upstream kinases for activation and inactivation by depho- kinase on threonine 308 (T308) and serine 473 (S473).3,5 sphorylation by protein phosphatases. Phosphorylated AKT is A recent study has indicated that phosphorylation of AKT at commonly found in acute myeloid leukemia (AML) and confers 9 an unfavorable prognosis. Understanding the relative impor- T308 but not S473 is a poor prognostic factor for AML patients. tance of upstream kinases and AKT phosphatase in the The regulation of signal transduction is a dynamic process activation of AKT is relevant for the therapeutic targeting of dependent on the balance between phosphorylation by protein this signaling axis in AML. The B55a subunit of protein kinases and dephosphorylation by protein phosphatases.10,11 phosphatase 2A (PP2A) has been implicated in AKT depho- Aberrant activation of AKT in a leukemia cell could result either sphorylation, but its role in regulating AKT in AML is unknown.
    [Show full text]
  • Loss of PPP2R2A Inhibits Homologous Recombination DNA Repair and Predicts Tumor Sensitivity to PARP Inhibition
    Published OnlineFirst October 18, 2012; DOI: 10.1158/0008-5472.CAN-12-1667 Cancer Molecular and Cellular Pathobiology Research Loss of PPP2R2A Inhibits Homologous Recombination DNA Repair and Predicts Tumor Sensitivity to PARP Inhibition Peter Kalev1, Michal Simicek1, Iria Vazquez1, Sebastian Munck1, Liping Chen2, Thomas Soin1, Natasha Danda1, Wen Chen2, and Anna Sablina1 Abstract Reversible phosphorylation plays a critical role in DNA repair. Here, we report the results of a loss-of-function screen that identifies the PP2A heterotrimeric serine/threonine phosphatases PPP2R2A, PPP2R2D, PPP2R5A, and PPP2R3C in double-strand break (DSB) repair. In particular, we found that PPP2R2A-containing complexes directly dephosphorylated ATM at S367, S1893, and S1981 to regulate its retention at DSB sites. Increased ATM phosphorylation triggered by PPP2R2A attenuation dramatically upregulated the activity of the downstream effector kinase CHK2, resulting in G1 to S-phase cell-cycle arrest and downregulation of BRCA1 and RAD51. In tumor cells, blocking PPP2R2A thereby impaired the high-fidelity homologous recombination repair pathway and sensitized cells to small-molecule inhibitors of PARP. We found that PPP2R2A was commonly downregulated in non–small cell lung carcinomas, suggesting that PPP2R2A status may serve as a marker to predict therapeutic efficacy to PARP inhibition. In summary, our results deepen understanding of the role of PP2A family phosphatases in DNA repair and suggest PPP2R2A as a marker for PARP inhibitor responses in clinic. Cancer Res; 72(24); 1–11. Ó2012 AACR. Introduction during the DNA repair process (3, 4), suggesting that phos- Genome stability is essential for the prevention of undue phatases may serve not only as negative regulators but also as cellular death and cancer development.
    [Show full text]
  • PPP2R2C Loss Promotes Castration-Resistance and Is Associated with Increased Prostate Cancer-Specific Mortality
    Published OnlineFirst March 14, 2013; DOI: 10.1158/1541-7786.MCR-12-0710 Molecular Cancer Cell Death and Survival Research PPP2R2C Loss Promotes Castration-Resistance and Is Associated with Increased Prostate Cancer-Specific Mortality Eric G. Bluemn1,2, Elysia Sophie Spencer1, Brigham Mecham2,3, Ryan R. Gordon2, Ilsa Coleman2, Daniel Lewinshtein2,5, Elahe Mostaghel1,2, Xiaotun Zhang1, James Annis1,4, Carla Grandori1,2,4, Christopher Porter5, and Peter S. Nelson1,2 Abstract Metastatic prostate cancers generally rely on androgen receptor (AR) signaling for growth and survival, even following systemic androgen-deprivation therapy (ADT). However, recent evidence suggests that some advanced prostate cancers escape ADT by using signaling programs and growth factors that bypass canonical AR ligand- mediated mechanisms. We used an in vitro high-throughput RNA interference (RNAi) screen to identify pathways in androgen-dependent prostate cancer cell lines whose loss-of-function promotes androgen ligand-independent growth. We identified 40 genes where knockdown promoted proliferation of both LNCaP and VCaP prostate cancer cells in the absence of androgen. Of these, 14 were downregulated in primary and metastatic prostate cancer, including two subunits of the protein phosphatase 2 (PP2A) holoenzyme complex: PPP2R1A, a structural subunit with known tumor-suppressor properties in several tumor types; and PPP2R2C, a PP2A substrate-binding regulatory subunit that has not been previously identified as a tumor suppressor. We show that loss of PPP2R2C promotes androgen ligand depletion-resistant prostate cancer growth without altering AR expression or canonical AR-regulated gene expression. Furthermore, cell proliferation induced by PPP2R2C loss was not inhibited by the AR antagonist MDV3100, indicating that PPP2R2C loss may promote growth independently of known AR- mediated transcriptional programs.
    [Show full text]
  • A Cancer-Associated Missense Mutation in PP2A-Aα Increases Centrosome Clustering During Mitosis
    bioRxiv preprint doi: https://doi.org/10.1101/570978; this version posted March 7, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. A cancer-associated missense mutation in PP2A-Aα increases centrosome clustering during mitosis Noelle V. Antao1, 2, Marina Marcet-Ortega2, Paolo Cifani3, Alex Kentsis3, Emily A. Foley2* 1. Program in Biochemistry and Structural Biology, Cell and Developmental Biology, and Molecular Biology, Weill Cornell Medicine Graduate School of Medical Sciences, Cornell University, New York, NY 10065, USA. 2. Cell Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA. 3. Molecular Pharmacology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA *corresponding author: [email protected] Running title: PP2A inactivation enhances centrosome clustering bioRxiv preprint doi: https://doi.org/10.1101/570978; this version posted March 7, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Abstract A single incidence of whole-genome doubling (WGD) is common early in tumorigenesis. In addition to increasing ploidy, WGD doubles centrosome number. In the ensuing mitoses, excess centrosomes form a multipolar spindle, resulting in a lethal multipolar cell division. To survive, cells must cluster centrosomes into two poles to allow a bipolar cell division. Cancer cells are typically more proficient at centrosome clustering than untransformed cells, but the mechanism behind increased clustering ability is not well understood. Heterozygous missense mutations in PPP2R1A, which encodes the alpha isoform of the A-subunit of protein phosphatase 2A (PP2A-Aα), positively correlate with WGD.
    [Show full text]
  • Fusion of the Tumor-Suppressor Gene CHEK2 and the Gene for the Regulatory Subunit B of Protein Phosphatase 2 PPP2R2A in Childhood Teratoma1
    RESEARCH ARTICLE Neoplasia . Vol. 8, No. 5, May 2006, pp. 413 – 418 413 www.neoplasia.com Fusion of the Tumor-Suppressor Gene CHEK2 and the Gene for the Regulatory Subunit B of Protein Phosphatase 2 PPP2R2A in Childhood Teratoma1 Yuesheng Jin*, Fredrik Mertens*, Carl-Magnus Kullendorff y and Ioannis Panagopoulos* *Department of Clinical Genetics, Lund University Hospital, 221 85 Lund, Sweden; yDepartment of Pediatric Surgery, Lund University Hospital, 221 85 Lund, Sweden Abstract We characterized the molecular genetic consequences aggressive subtypes of germ cell tumors, which typically have of a balanced chromosome translocation t(8;22)(p21; aneuploid karyotypes with a gain of the short arm of chro- q12), which occurred as the sole cytogenetic aberration mosome 12 as the most common feature, mature teratomas in short-term cultured cells from an intrathoracic ma- often display normal karyotype on cytogenetic analysis, and no ture teratoma in a 15-year-old girl. Fluorescence in situ consistent pattern of chromosomal gains or losses has been hybridization and reverse transcription–polymerase disclosed by comparative genomic hybridization [3,4]. Further- chain reaction disclosed that t(8;22) resulted in the more, neither in mature teratomas nor in germ cell tumors, in fusion of the genes PPP2R2A and CHEK2,withan general, has any recurrent, acquired, tumor-specific, balanced inserted fragment belonging to class I endogenous chromosome rearrangement been detected. Recently, how- retrovirus–related sequences at the junction. Sequenc- ever, a constitutional t(12;15)(q13;q25), resulting in the fusion ing of the two genes did not reveal any additional mu- of the genes SENP1 and MESDC2, was identified in a patient tation.
    [Show full text]