Journal: Developmental Cell

Article Title: Reductional Meiosis I Chromosome Segregation Is Established by Coordination of Key Meiotic Kinases

doi: 10.1016/j.devcel.2019.04.003

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Article Snippet: PP1 , Toronto Research Chemicals , A602980.

Techniques: Recombinant, Phospho-proteomics, Ligation, Expressing, Mass Spectrometry, Generated, Software, Microscopy

Journal: Cell Reports

Article Title: Fission Yeast NDR/LATS Kinase Orb6 Regulates Exocytosis via Phosphorylation of the Exocyst Complex

doi: 10.1016/j.celrep.2019.01.027

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Article Snippet: To inhibit Orb6 kinase activity in vivo , 3-BrB-PP1 (Toronto Research Chemicals; A602985), 3-MB-PP1 (Toronto Research Chemicals; A602960), 1-NM-PP1 (Toronto Research Chemicals; A603003) dissolved in methanol were each used at a final concentration of 30 μM.

Techniques: Affinity Purification, Virus, Recombinant, Labeling, Fluorescence, Plasmid Preparation, Over Expression, Expressing, Software, Protein Purification, Centrifugation, Mass Spectrometry, Membrane

Journal: Developmental Cell

Article Title: Reductional Meiosis I Chromosome Segregation Is Established by Coordination of Key Meiotic Kinases

doi: 10.1016/j.devcel.2019.04.003

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Article Snippet: 1-NM-PP1 , Toronto Research Chemicals , A603004.

Techniques: Recombinant, Phospho-proteomics, Ligation, Expressing, Mass Spectrometry, Generated, Software, Microscopy

Journal: BMC Plant Biology

Article Title: Modulation of plant growth in vivo and identification of kinase substrates using an analog-sensitive variant of CYCLIN-DEPENDENT KINASE A;1

doi: 10.1186/s12870-016-0900-7

Figure Lengend Snippet: Generation and characterization of an analog-sensitive variant of CDKA;1. a Sketch of an analog-sensitive kinase variant ( right ) that has an enlarged ATP-binding pocket in comparison with the wild-type version ( left ) through exchanging a ‘gatekeeper’ amino acid (position in magenta), typically a large amino acid, in the wild-type version with a small one such as Gly. An analog-sensitive kinase can use regular ATP but also bulky derivatives that cannot be used by the wild-type variant as a phosphate donor (see also below). b Computed 3D structure of the ATP binding pocket of CDKA;1. In magenta, the space occupied at the bottom of the pocket by the gatekeeper amino acid Phenylalanine (Phe/F) 80 in the wild-type kinase that will be enlarged by the F80 to Glycine (Gly/G) mutation. c Structure of adenosine triphosphate (ATP). d Structure of the bulky-ATP derivate N 6 -(2-Phenylethyl)adenosine-5′-O-triphosphate (N6-PhEt-ATP). e In vitro kinase assay with wild-type and the analog-sensitive CDKA;1 (CDKA;1 F80G ) kinases using CYCD3;1 as a cyclin partner and histone H1 as a generic substrate. First lane from the top, protein blotting reveals equal amounts of CDKA;1 proteins in the reaction. Second lane, kinase assays with [γ- 32 P]-ATP as a phosphate donor. Forth lane, kinase assays with [γ- 32 P]-N6-PhEt-ATP as a phosphate donor. Proteins were subjected to SDS-PAGE after the kinase reaction and stained with Coomassie brilliant blue R-250 demonstrating equal loading of the substrate, lane three and five from the top. Abbreviations: p-H1 for radio-labeled histone H1 resulting from kinase assays with radio-labeled ATP, H1 for histone H1. f Structure of the broad band kinase inhibitor 4-amino-1- tert -butyl-3-phenylpyrazolo[3,4- d ]pyrimidine (PP1) on the left and the bulky analogs 4-amino-1- tert -butyl-3-(1′-naphthyl)pyrazolo[3,4- d ]pyrimidine (1-NA-PP1) in the middle as well as 4-amino-1- tert -butyl-3-(1′-naphthylmethyl)pyrazolo[3,4- d ]pyrimidine (1-NM-PP1) on the right. g In vitro kinase assay with wild-type and the analog-sensitive CDKA;1 (CDKA;1 F80G ) kinases using CYCD3;1 as a cyclin partner and histone H1 as a generic substrate. Inhibition of wild-type ( left ) and the analog-sensitive CDKA;1 ( right ) kinases with 0, 1, and 10 μM of the PP1 derivative 1-NM-PP1. Proteins were subjected to SDS-PAGE after the kinase reaction with [γ- 32 P]-ATP as a phosphate donor and stained with Coomassie brilliant blue R-250 demonstrating equal loading of the substrate. Mock was treated with 0.1 % (v/v) DMSO, the solvent of 1-NM-PP1. Abbreviations : p-H1 for radio-labeled histone H1 resulting from kinase assays with radio-labeled ATP, H1 for histone H1. Chemical structures in this figure were drawn with MarvinSketch, version 5.0.02 (ChemAxon, Hungary)

Article Snippet: The PP1 analogs (4-amino-1- tert -butyl-3-(1′-naphthyl)pyrazolo[3,4- d ]pyrimidine (1-NA-PP1) and 1-NM-PP1; Toronto Research Chemicals Inc.) were prepared as 10 mM stock solution in DMSO.

Techniques: Variant Assay, Binding Assay, Comparison, Mutagenesis, In Vitro, Kinase Assay, SDS Page, Staining, Labeling, Inhibition, Solvent

Journal: BMC Plant Biology

Article Title: Modulation of plant growth in vivo and identification of kinase substrates using an analog-sensitive variant of CYCLIN-DEPENDENT KINASE A;1

doi: 10.1186/s12870-016-0900-7

Figure Lengend Snippet: Modulation of plant growth in vivo. a Wild-type control plants grown for 2 weeks on MS plates containing the solvent DMSO and no bulky kinase inhibitor. Scale bar: 4 mm. b Wild-type plants grown for 2 weeks on MS plates supplemented with 100 μM 1-NA-PP1 die. Scale bar: 4 mm. c Wild-type plants grown for 2 weeks on MS plates supplemented with 100 μM 1-NM-PP1 are smaller than wild-type plants grown without the inhibitor but survive. Scale bar: 4 mm. d cdka;1 −/− PRO CDKA;1 :CDKA;1 F80G ( cdka;1-as ) grown for 2 weeks on MS plates containing the solvent DMSO and no bulky kinase inhibitor are slight reduced in their size in comparison with the wild-type control plants, see also Fig. . Scale bar: 4 mm. e cdka;1-as grown for 2 weeks on MS plates supplemented with 100 μM 1-NM-PP1 is severely compromised with arrested root development. Scale bar: 4 mm. f A homozygous cdka;1 −/− seedling grown on a MS plate for 2 weeks after germination shows the typical phenotype of loss of CDKA;1 function with halted root development and only a few and tiny leaves being formed. Scale bar: 1 mm. g Root length measurement of the seedlings of wild type (Col-0) and cdka;1-as ( as ) grown on MS plates supplemented with 10 μM 1-NM-PP1 for 7 days after germination. Error bars represent the SE. A statistical significant change between the mock and 1-NM-PP1 treatment is marked by an asterisk above the bar (t-test, P = 0.0002 <0.05)

Article Snippet: The PP1 analogs (4-amino-1- tert -butyl-3-(1′-naphthyl)pyrazolo[3,4- d ]pyrimidine (1-NA-PP1) and 1-NM-PP1; Toronto Research Chemicals Inc.) were prepared as 10 mM stock solution in DMSO.

Techniques: In Vivo, Control, Solvent, Comparison