• 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.

      Tay, Ye Day; Patel, Avinash; Kaemena, Daniel F; Hagan, Iain M (2013-11-01)
      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.