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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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References

REFERENCES

Brun, R. & Schonenberger, M. (1979). Cultivation and in vitro cloning of procyclic culture forms of T. brucei ina semi-defined medium. Acta Tropica 36, 289–92.Google Scholar
Cassel, D., Shoubi, S., Glusman, G., Cukierman, E., Rotman, M. & Zilberstein, D. (1991). Leishmania donovani: Characterisation of a 38 kDa membrane protein that cross reacts with the mammalian G-protein Transducin. Experimental Parasitology 72, 411–17.CrossRefGoogle ScholarPubMed
Cooper, J. A., Sefton, B. M. & Hunter, T. (1983). Detection and quantification of phosphotyrosine in proteins. Methods in Enzymology 99, 387–402.CrossRefGoogle ScholarPubMed
Davis, C. E., Colmeraner, M. E. H., Kim, C.-H., Matthews, B. & Guiney, D. (1989). myc-Homologues in Trypanosoma brucei. Microbial Pathology 7, 45–53.CrossRefGoogle ScholarPubMed
Feder, D. & Bishop, J. M. (1990). Purification and enzymatic characterisation of pp60c-src from human platelets. Journal of Biological Chemistry 265, 8205–11.CrossRefGoogle ScholarPubMed
Gomez, M. L., Erijman, L., Arauzo, S., Torres, H. N. & Tellez-Inon, M. T. (1989). Protein kinase C in T. cruzi epimastigote forms: partial purification and characterisation. Molecular and Biochemical Parasitology 36, 101–8.CrossRefGoogle Scholar
Hide, G., Gray, A., Harrison, C. M. & Tait, A. (1989). Identification of an epidermal growth factor receptor homologue in trypanosomes. Molecular and Biochemical Parasitology 36, 51–60.CrossRefGoogle ScholarPubMed
Hide, G., Gray, A., Harrison, C. M. & Tait, A. (1990). Identification of an EGF receptor homologue in trypanosomes. In Parasites: Molecular Biology, Drug and Vaccine Design, vol. 131 (ed. Agabian, N. & Cerami, A.), pp. 213–224. New York: Wiley-Liss.Google Scholar
Hide, G., Tait, A. & Keith, K. (1990). Characterisation of Trypanosoma brucei protein kinases and a growth factor receptor. Biochemical Society Transactions 18, 733–5.CrossRefGoogle Scholar
Keith, K., Hide, G. & Tait, A. (1990). Characterisation of protein kinase C like activities in Trypanosoma brucei. Molecular and Biochemical Parasitology 43, 107–16.CrossRefGoogle ScholarPubMed
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, London 227, 680–5.CrossRefGoogle ScholarPubMed
Lanham, S. M. & Godfrey, D. G. (1970). Isolation of salivarian trypanosomes from man and other animals using DEAE-cellulose. Experimental Parasitology 28, 521–34.CrossRefGoogle ScholarPubMed
Parsons, M., Valentine, M., Deans, J., Schieven, G. L. & Ledbetter, J. A. (1991). Distinct patterns of tyrosine phosphorylation during the life cycle of Trypanosoma brucei. Molecular and Biochemical Parasitology 44, 241–9.CrossRefGoogle Scholar
Towbin, H., Staehelin, T. & Gordon, J. (1979). Electrophoretic transfer of proteins from polyacrylamide gels onto nitrocellulose: procedure and some applications. Proceedings of the National Academy of Sciences, USA 76, 4350–4.CrossRefGoogle Scholar
Walter, R. D. (1978). Multiple protein kinases from T. gambiense. Hoppe-Seylers Zeitschrift für physiologische Chemie 359, 601–6.Google Scholar
Wheeler, E. & Shapiro, S. Z. (1992). Evidence of tyrosine kinase activity in the protozoan parasite. Trypanosoma brucei. Journal of Protozoology 39, 413–16.CrossRefGoogle Scholar