title: Mutation of a conserved residue enhances the sensitivity of analogue-sensitised kinases to generate a novel approach to the study of mitosis in fission yeast.
abstract: The chemical genetic strategy in which mutational enlargement of the ATP-binding site sensitises of a protein kinase to bulky ATP analogues has proved to be an elegant tool for the generation of conditional analogue-sensitive kinase alleles in a variety of model organisms. Here, we describe a novel substitution mutation in the kinase domain that can enhance the sensitivity of analogue-sensitive kinases. Substitution of a methionine residue to phenylalanine in the +2 position after HRDLKxxN motif of the subdomain VIb within the kinase domain markedly increased the sensitivities of the analogue-sensitive kinases to ATP analogues in three out of five S. pombe kinases (i.e. Plo1, Orb5 and Wee1) that harbor this conserved methionine residue. Kinome alignment established that a methionine residue is found at this site in 5-9% of kinases in key model organisms, suggesting that a broader application of this structural modification may enhance ATP analogue sensitivity of analogue-sensitive kinases in future studies. We also show that the enhanced sensitivity of the wee1.as8 allele in a cdc25.22 background can be exploited to generate highly synchronised mitotic and S phase progression at 36°C. Proof-of-principle experiments show how this novel synchronisation technique will prove of great use in the interrogation of the mitotic or S-phase functions through temperature sensitivity mutation of molecules of interest in fission yeast.
authors.affiliation: CRUK Cell Division Group, Paterson Institute for Cancer Research, Wilmslow Road, Manchester M20 4BX, UK.
authors.forename: Ye-Dee
authors.initials: YD
authors.lastname: Tay
authors.forename: Avinash
authors.initials: A
authors.lastname: Patel
authors.forename: Daniel F
authors.initials: DF
authors.lastname: Kaemena
authors.forename: Iain M
authors.initials: IM
authors.lastname: Hagan
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D019943:Amino Acid Substitution; D047390:Casein Kinase II / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017124:Conserved Sequence; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism; D008715:Methionine / Q000235:genetics / Q000378:metabolism; D008938:Mitosis; D020125:Mutation, Missense; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D010649:Phenylalanine / Q000235:genetics / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000235:genetics / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D012568:Schizosaccharomyces / Q000166:cytology / Q000201:enzymology* / Q000235:genetics; D029702:Schizosaccharomyces pombe Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
chemical_list: D018797:Cell Cycle Proteins; D004791:Enzyme Inhibitors; D009687:Nuclear Proteins; D029702:Schizosaccharomyces pombe Proteins; D010649:Phenylalanine; D000255:Adenosine Triphosphate; D008715:Methionine; C068689:wee1 protein, S pombe; D011505:Protein-Tyrosine Kinases; D047390:Casein Kinase II; C501407:Plo1 protein, S pombe; D017346:Protein Serine-Threonine Kinases; C479661:casein kinase II, S pombe; C570820:Cdc25 protein, S pombe; D010749:Phosphoprotein Phosphatases
keywords: Analogue-sensitive kinase; Cdc25; Mitosis; S. pombe; Wee1