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PEDS Advance Access published online on January 10, 2009
Protein Engineering Design and Selection, doi:10.1093/protein/gzn078
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© The Author 2009. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Mutations at serine 37 in mouse guanylate kinase confer resistance to 6-thioguanine

Andressa Ardiani1, Amanda Goyke2 and Margaret E. Black1,2,3

1School of Molecular Biosciences 2Department of Pharmaceutical Sciences, Washington State University, PO Box 646534, Pullman, WA 99164-6534, USA

3 To whom correspondence should be addressed. Department of Pharmaceutical Sciences, Washington State University, PO Box 646534, Pullman, WA 99164-6534, USA. E-mail: blackm{at}mail.wsu.edu

Guanylate kinase (GMK) is an essential nucleoside monophosphate kinase that catalyzes the phosphorylation of guanine-monophosphate (GMP) and dGMP to yield GDP and dGDP, respectively, important precursors for nucleotide synthesis. GMK is also responsible for the activation of 6-thioguanine (6-TG), a drug widely used as chemotherapeutic agent to treat leukemia. Several mechanisms of resistance to 6-TG have been reported but a subset of drug resistant cells cannot be explained by these mechanisms. We propose that mutations in GMK could result in drug resistance. Because cells require the presence of a functional GMK for viability, mutations that arise that lead to 6-TG resistance must retain activity toward GMP. We report three amino acid substitutions at serine 37 (S37) in mouse GMK that display activity toward GMP by conferring genetic complementation to a conditional GMK-deficient Escherichia coli and in enzyme assays. When 6-TG is included in complementation studies, cells expressing wild-type GMK are sensitive whereas all S37 mutants examined are able to effectively discriminate against 6-TG and display a drug resistance phenotype. Activity of the three S37 mutant enzymes toward clinically relevant concentrations of 6-TGMP is undetectable. Mutations in GMK, therefore, represent a previously undescribed mechanism for 6-TG resistance.

Keywords: 6-thioguanine/drug resistance/guanylate kinase/mutagenesis/selection

Received July 15, 2008; revised October 21, 2008; accepted November 21, 2008.


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