Regulators of Trypanosoma brucei cell cycle progression and differentiation identified using a kinome-wide RNAi screen

Jones, N. G., Thomas, E. B., Brown, E., Dickens, N. J. , Hammarton, T. C. and Mottram, J. C. (2014) Regulators of Trypanosoma brucei cell cycle progression and differentiation identified using a kinome-wide RNAi screen. PLoS Pathogens, 10(1), e1003886. (doi: 10.1371/journal.ppat.1003886)

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Publisher's URL: http://dx.doi.org/10.1371/journal.ppat.1003886

Abstract

The African trypanosome, Trypanosoma brucei, maintains an integral link between cell cycle regulation and differentiation during its intricate life cycle. Whilst extensive changes in phosphorylation have been documented between the mammalian bloodstream form and the insect procyclic form, relatively little is known about the parasite's protein kinases (PKs) involved in the control of cellular proliferation and differentiation. To address this, a T. brucei kinome-wide RNAi cell line library was generated, allowing independent inducible knockdown of each of the parasite's 190 predicted protein kinases. Screening of this library using a cell viability assay identified ≥42 PKs that are required for normal bloodstream form proliferation in culture. A secondary screen identified 24 PKs whose RNAi-mediated depletion resulted in a variety of cell cycle defects including in G1/S, kinetoplast replication/segregation, mitosis and cytokinesis, 15 of which are novel cell cycle regulators. A further screen identified for the first time two PKs, named repressor of differentiation kinase (RDK1 and RDK2), depletion of which promoted bloodstream to procyclic form differentiation. RDK1 is a membrane-associated STE11-like PK, whilst RDK2 is a NEK PK that is essential for parasite proliferation. RDK1 acts in conjunction with the PTP1/PIP39 phosphatase cascade to block uncontrolled bloodstream to procyclic form differentiation, whilst RDK2 is a PK whose depletion efficiently induces differentiation in the absence of known triggers. Thus, the RNAi kinome library provides a valuable asset for functional analysis of cell signalling pathways in African trypanosomes as well as drug target identification and validation.

Item Type:Articles
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Mottram, Professor Jeremy and Dickens, Dr Nicholas and Brown, Miss Elaine and Jones, Mr Nathaniel and Hammarton, Dr Tansy
Authors: Jones, N. G., Thomas, E. B., Brown, E., Dickens, N. J., Hammarton, T. C., and Mottram, J. C.
College/School:College of Medical Veterinary and Life Sciences > Institute of Infection Immunity and Inflammation
Journal Name:PLoS Pathogens
Publisher:Public Library of Science
ISSN:1553-7366
ISSN (Online):1553-7374
Published Online:16 January 2014
Copyright Holders:Copyright © 2014 The Authors
First Published:First published in PLoS ONE 2014 10(1):e1003886
Publisher Policy:Reproduced under a Creative Commons License
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Project CodeAward NoProject NamePrincipal InvestigatorFunder's NameFunder RefLead Dept
371796The Wellcome Centre for Molecular Parasitology ( Core Support )Andrew WatersWellcome Trust (WELLCOME)085349/Z/08/ZIII - PARASITOLOGY