TRYPANOSOMA-BRUCEI - CHARACTERIZATION OF PROTEIN-KINASES THAT ARE CAPABLE OF AUTOPHOSPHORYLATION IN-VITRO

Autophosphorylation by protein kinases has been implicated as an impor tant control mechanism in signal transduction and growth regulatory pa thways in mammalian cells. We have set out to investigate whether any such autophosphorylating protein kinase activities can be found in Try panosoma brucei. In order to do this, we have developed a system for c haracterizing such protein kinase activities using an in vitro assay. This assay was carried out by fractionation of trypanosome lysates usi ng isoelectric focusing gel electrophoresis followed by incubation of the gel in gamma(32)P-labelled nucleotide triphosphate and subsequent autoradiography. We have identified two classes of autophosphorylating protein kinase activities. In the first class all were dependent on A TP as the phosphate donor substrate and were all found to have a molec ular size of 60 kDa. Differences in the activity of these protein kina ses were observed between the bloodstream and procyclic life-cyle stag es. Furthermore, the addition of mammalian epidermal growth factor to bloodstream stage lysates stimulated an additional activity. The secon d class of autophosphorylating protein kinases utilized GTP as the pho sphate donor and were all found to be 90 kDa in size. Stage-specific d ifferences were also observed in the activity of these protein kinases .