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

Reversible protein phosphorylation is essential for the regulation of numer ous cellular functions and differentiation. The haemo-flagellated parasitic protozoan Trypanosoma brucei, the causative agent for Africa trypanosomias is undergoes various stages of cellular differentiation during its digeneti c life cycle. A complete cDNA of a unique serine/threonine phosphatase: typ e five (TbPP5) was cloned and characterized from T. brucei. TbPPS 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 ac id sequence showed 45-48% overall identity and 60-65% similarity with prote in phosphatase 5 's (PP5) from different species. Analysis of the primary s equence revealed that TbPPS contains three TPR motifs at the N-terminal reg ion (amino acid residues 7-107) while the phosphatase catalytic domain occu rs in the C-terminal region (amino acid residues 210-410). A TbPPS cDNA hyb ridized with a transcript of 2.5 kb which is present at similar levels in t he procyclic and the bloodstream forms. However, the level of expression of the TbPPS protein (52 kDa) detected by an antibody developed against a rec ombinant protein produced in E. coli was about 2-fold higher in the procycl ic than the bloodstream form. The TbPP5 transcript level gradually decrease d in cells grown in the logarithmic phase to the stationary phase in cultur e. Moreover, 18 h serum starvation of the procyclic forms decreased the lev el of the specific transcript about 3-fold suggesting that this: protein ma y play a role during the active growth phase of the organism. The recombina nt protein showed phosphatase activity which was stimulated about 2.6-fold by arachidonic acid with half-maximal activity at 75 muM Indirect immuno-fl uoresence 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 s uch as T. brucei. (C) 2001 Elsevier Science B.V. All rights reserved.