Nuclear DBF-2-related kinases are essential regulators of cytokinesis in bloodstream stage Trypanosoma brucei

Ma, J., Benz, C., Grimaldi, R., Stockdale, C., Wyatt, P., Frearson, J. and Hammarton, T. C. (2010) Nuclear DBF-2-related kinases are essential regulators of cytokinesis in bloodstream stage Trypanosoma brucei. Journal of Biological Chemistry, 285(20), pp. 15356-15368. (doi:10.1074/jbc.M109.074591)

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Publisher's URL: http://dx.doi.org/10.1074/jbc.M109.074591

Abstract

Nuclear DBF-2-related (NDR) kinases are essential regulators of cell cycle progression, growth, and development in many organisms and are activated by the binding of an Mps One Binder (MOB) protein partner, autophosphorylation, and phosphorylation by an upstream STE20 family kinase. In the protozoan parasite, Trypanosoma brucei, the causative agent of human African trypanosomiasis, the NDR kinase, PK50, is expressed in proliferative life cycle stages and was shown to complement a yeast NDR kinase mutant cell line. However, the function of PK50 and a second NDR kinase, PK53, in T. brucei has not been determined to date, although trypanosome MOB1 is known to be essential for cytokinesis, suggesting the NDR kinases may also be involved in this process. Here, we show that specific depletion of PK50 or PK53 from bloodstream stage trypanosomes resulted in the rapid accumulation of cells with two nuclei and two kinetoplasts, indicating that cytokinesis was specifically inhibited. This led to a deregulation of the cell cycle and cell death and provides genetic validation of these kinases as potential novel drug targets for human African trypanosomiasis. Recombinant active PK50 and PK53 were produced and biochemically characterized. Both enzymes autophosphorylated, were able to trans-phosphorylate generic kinase substrates in vitro, and were active in the absence of phosphorylation by an upstream kinase. Additionally, both enzymes were active in the absence of MOB1 binding, which was also demonstrated to likely be a feature of the kinases in vivo. Biochemical characterization of recombinant PK50 and PK53 has revealed key kinetic differences between them, and the identification of in vitro peptide substrates in this study paves the way for high throughput inhibitor screening of these kinases.

Item Type:Articles
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Benz, Dr Corinna and Hammarton, Dr Tansy and Stockdale, Dr Christopher and Wyatt, Prof Paul and Ma, Dr Jiangtao
Authors: Ma, J., Benz, C., Grimaldi, R., Stockdale, C., Wyatt, P., Frearson, J., and Hammarton, T. C.
Subjects:Q Science > QR Microbiology
College/School:College of Medical Veterinary and Life Sciences > Institute of Infection Immunity and Inflammation
Journal Name:Journal of Biological Chemistry
Journal Abbr.:J Biol Chem.
Publisher:American Society for Biochemistry and Molecular Biology, Inc.
ISSN:0021-9258
ISSN (Online):1083-351X
Published Online:15 March 2010
Copyright Holders:Copyright © 2010 The American Society for Biochemistry and Molecular Biology, Inc.
First Published:First published in Journal of Biological Chemistry 285(20):15356-15368
Publisher Policy:Reproduced under a Creative Commons License
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Project CodeAward NoProject NamePrincipal InvestigatorFunder's NameFunder RefLead Dept
403591Regulation of cytokinesis in trypanosoma bruceiTansy HammartonMedical Research Council (MRC)G120/1001Infection Immunity and Inflammation Life Sciences
493121The NDR kinase pathway in Trypanosoma bruceiTansy HammartonMedical Research Council (MRC)G0900239Infection Immunity and Inflammation Life Sciences