Cloning and characterization of a novel serine/threonine protein phosphatase type 5 from Trypanosoma brucei.

Reversible protein phosphorylation is essential for the regulation of numerous cellular functions and differentiation. The haemo-flagellated parasitic protozoan Trypanosoma brucei, the causative agent for African trypanosomiasis undergoes various stages of cellular differentiation during its digenetic life cycle. A complete cDNA of a unique serine/threonine phosphatase type five (TbPP5) was cloned and characterized from T. brucei. TbPP5 contains an open reading frame of 1416 bp that encodes a protein of about 53 kDa and exists as a single copy gene in the T. brucei genome. The deduced amino acid sequence showed 45-48% overall identity and 60-65% similarity with protein phosphatase 5's (PP5) from different species. Analysis of the primary sequence revealed that TbPP5 contains three TPR motifs at the N-terminal region (amino acid residues 7-107) while the phosphatase catalytic domain occurs in the C-terminal region (amino acid residues 210-410). A TbPP5 cDNA hybridized with a transcript of 2.5 kb which is present at similar levels in the procyclic and the bloodstream forms. However, the level of expression of the TbPP5 protein (52 kDa) detected by an antibody developed against a recombinant protein produced in E. coli was about 2-fold higher in the procyclic than the bloodstream form. The TbPP5 transcript level gradually decreased in cells grown in the logarithmic phase to the stationary phase in culture. Moreover, 18 h serum starvation of the procyclic forms decreased the level of the specific transcript about 3-fold suggesting that this protein may play a role during the active growth phase of the organism. The recombinant protein showed phosphatase activity which was stimulated about 2.6-fold by arachidonic acid with half-maximal activity at 75 microM. Indirect immuno-fluorescence of permeabilized cells revealed that the protein is localized in the cytosol and the nucleus This is the first report for the identification of a type 5 serine/threonine protein phosphatase in an ancient eukaryote such as T. brucei.