title: Optimization of the analogue-sensitive Cdc2/Cdk1 mutant by in vivo selection eliminates physiological limitations to its use in cell cycle analysis.
abstract: Analogue-sensitive (as) mutants of kinases are widely used to selectively inhibit a single kinase with few off-target effects. The analogue-sensitive mutant cdc2-as of fission yeast (Schizosaccharomyces pombe) is a powerful tool to study the cell cycle, but the strain displays meiotic defects, and is sensitive to high and low temperature even in the absence of ATP-analogue inhibitors. This has limited the use of the strain for use in these settings. Here, we used in vivo selection for intragenic suppressor mutations of cdc2-as that restore full function in the absence of ATP-analogues. The cdc2-asM17 underwent meiosis and produced viable spores to a similar degree to the wild-type strain. The suppressor mutation also rescued the sensitivity of the cdc2-as strain to high and low temperature, genotoxins and an anti-microtubule drug. We have used cdc2-asM17 to show that Cdc2 activity is required to maintain the activity of the spindle assembly checkpoint. Furthermore, we also demonstrate that maintenance of the Shugoshin Sgo1 at meiotic centromeres does not require Cdc2 activity, whereas localization of the kinase aurora does. The modified cdc2-asM17 allele can be thus used to analyse many aspects of cell-cycle-related events in fission yeast.
authors.affiliation: Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Tokyo 113-0033, Japan Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Tokyo 113-0033, Japan.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Aoi
authors.affiliation: Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Tokyo 113-0033, Japan.
authors.forename: Shigehiro A
authors.initials: SA
authors.lastname: Kawashima
authors.affiliation: EPFL SV ISREC UPSIM SV2.1830, Station 19, Lausanne 1015, Switzerland.
authors.forename: Viesturs
authors.initials: V
authors.lastname: Simanis
authors.affiliation: Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Tokyo 113-0033, Japan Laboratory of Cell Responses, National Institute for Basic Biology, Nishigonaka 38, Myodaiji, Okazaki, Aichi 444-8585, Japan.
authors.forename: Masayuki
authors.initials: M
authors.lastname: Yamamoto
authors.affiliation: Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Tokyo 113-0033, Japan PRESTO, Japan Science and Technology Agency, Gobancho, Chiyoda-ku, Tokyo 102-0076, Japan Laboratory of Cytoskeletal Logistics, Department of Life Science and Medical Bioscience, Graduate School of Advanced Science and Engineering, Waseda University, TWIns, 2-2 Wakamatsucho, Shinjuku, Tokyo 162-8480, Japan masasato@waseda.jp.
authors.forename: Masamitsu
authors.initials: M
authors.lastname: Sato
mesh_terms: D016203:CDC2 Protein Kinase / Q000235:genetics* / Q000378:metabolism; D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D012568:Schizosaccharomyces / Q000166:cytology* / Q000235:genetics* / Q000378:metabolism; D029702:Schizosaccharomyces pombe Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: D018797:Cell Cycle Proteins; D029702:Schizosaccharomyces pombe Proteins; D016203:CDC2 Protein Kinase
keywords: analogue-sensitive mutant; cell cycle; chemical genetics; cyclin-dependent kinase; fission yeast