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

Tay, Ye-Dee, Patel, Avinash, Kaemena, Daniel F. and Hagan, Iain M. 2013. Mutation of a conserved residue enhances sensitivity of analogue sensitized kinases to generate a novel approach for mitotic studies in fission yeast. Journal of Cell Science 126 (21) , 5052–5061. 10.1242/jcs.135301

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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.

Item Type:	Article
Date Type:	Publication
Status:	Published
Schools:	Biosciences
Publisher:	The Company of Biologists
ISSN:	0021-9533
Funders:	Cancer Research UK
Date of Acceptance:	30 July 2013
Last Modified:	18 Jul 2024 10:45
URI:	https://orca.cardiff.ac.uk/id/eprint/170129

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