Mutation of a conserved residue enhances sensitivity of analogue sensitized kinases to generate a novel approach for mitotic studies in fission yeast

Abstract

The chemical genetic strategy in which mutational enlargement of the ATP binding site sensitizes of a protein kinase to bulky ATP analogues has proved to be an elegant tool for the generation of conditional, analogue-sensitive (as), kinase alleles in a variety of model organisms. Here we described a novel substitution mutation in the kinase domain that can enhance the sensitivity of as kinase. 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 as kinases to ATP analogue 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 of 5–9% of kinases in key model organisms, suggesting that a broader application of this structural modification may enhance ATP analogue sensitivity of as 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 synchronized mitotic and S phase progression at 36°C. Proof-of-principle experiments show how this novel synchronization technique will prove of great utility in the interrogation of the mitotic or S-phase functions through temperature sensitivity mutation of molecules of interest in fission yeast.