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Trypanosoma brucei: characterization of protein kinases that are capable of autophosphorylation in vitro

Published online by Cambridge University Press:  06 April 2009

G. Hide ,
T. Graham ,
N. Buchanan ,
A. Tait  and
K. Keith
G. Hide
Affiliation:
Wellcome Unit of Molecular Parasitology, Department of Veterinary Parasitology, Glasgow University Veterinary School, Bearsden Road, Glasgow G61 1QH
T. Graham
Affiliation:
Wellcome Unit of Molecular Parasitology, Department of Veterinary Parasitology, Glasgow University Veterinary School, Bearsden Road, Glasgow G61 1QH
N. Buchanan
Affiliation:
Wellcome Unit of Molecular Parasitology, Department of Veterinary Parasitology, Glasgow University Veterinary School, Bearsden Road, Glasgow G61 1QH
A. Tait
Affiliation:
Wellcome Unit of Molecular Parasitology, Department of Veterinary Parasitology, Glasgow University Veterinary School, Bearsden Road, Glasgow G61 1QH
K. Keith
Affiliation:
Wellcome Unit of Molecular Parasitology, Department of Veterinary Parasitology, Glasgow University Veterinary School, Bearsden Road, Glasgow G61 1QH
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Summary

Autophosphorylation by protein kinases has been implicated as an important control mechanism in signal transduction and growth regulatory pathways in mammalian cells. We have set out to investigate whether any such autophosphorylating protein kinase activities can be found in Trypanosoma brucei. In order to do this, we have developed a system for characterizing such protein kinase activities using an in vitro assay. This assay was carried out by fractionation of trypanosome lysates using isoelectric focusing gel electrophoresis followed by incubation of the gel in γ32P-labelled nucleotide triphosphate and subsequent autoradiography. We have identified two classes of autophosphorylating protein kinase activities. In the first class all were dependent on ATP as the phosphate donor substrate and were all found to have a molecular size of 60 kDa. Differences in the activity of these protein kinases were observed between the bloodstream and procyclic life-cyle stages. Furthermore, the addition of mammalian epidermal growth factor to bloodstream stage lysates stimulated an additional activity. The second class of autophosphorylating protein kinases utilized GTP as the phosphate donor and were all found to be 90 kDa in size. Stage-specific differences were also observed in the activity of these protein kinases.

Keywords

KinetoplastidaTrypanosoma bruceiprotein kinaseprotein phosphorylationautophosphorylationepidermal growth factor
Type
Research Article
Information
Parasitology , Volume 108 , Issue 2 , February 1994 , pp. 161 - 166
Copyright
Copyright © Cambridge University Press 1994

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