Functional Characterization of the Serine/Threonine Protein Phosphatase 5 From Trypanosoma Brucei

Abstract

PP5 is a member of the PPP family of serine/threonine protein phosphatases and is present in all eukaryotes. We previously cloned and characterized a PP5 homologue from Trypanosoma brucei. Here, we synchronized the T. brucei procyclic form by hydroxyurea treatment and showed that TbPP5 expression is regulated during cell cycle progression. TbPP5 transcript and protein levels were maximal in the G1 phase of the cell cycle, and reduced about 3-fold in the G2/M phase. To further evaluate its function, TbPP5 expression was depleted in both procyclic and bloodstream forms of T. brucei by RNA interference. In the procyclic form, TbPP5 knockdown resulted in a moderate reduction in cell growth. However, in the bloodstream form, ablation of TbPP5 caused an 8-fold decrease in cell growth. Furthermore, TbPP5 overexpression conferred the ability of procyclic cells to grow in serum-deprived conditions suggesting that TbPP5 acts downstream of serum factor-induced growth in T. brucei. Taken together; these findings suggest that a serum factor (or factors) induces up-regulation of TbPP5 expression during the G1 phase, which is required for proper cell growth.

Protein phosphatase 5 is required for Hsp90 function during proteotoxic stresses in Trypanosoma brucei

“…Bloodstream cells were maintained in HMI-9 media supplemented with 10% heat-inactivated fetal bovine serum (Atlanta Biologicals), 10% serum plus (JRH Biosciences), and 2.5 μg/ml of geneticin (G418; Invitrogen). Procyclic cells were grown in SDM-79 media (JRH Biosciences), supplemented with 10% heat-inactivated fetal bovine serum, 15 μg/ml G418, and 50 μg/ml of hygromycin as described (Anderson et al 2006). Cells were treated with GA (Sigma-Aldrich) or 17 alylamino 17-demethoxygeldanamycin (17-AAG; Invivogen) at the final concentrations of 0-600 ng/ml for different time periods.…”

“…Generation of TbPP5 over-expressed and knocked down T. brucei cell lines TbPP5 over-expressing procyclic and the bloodstream form cell lines were generated as described (Anderson et al 2006) using the pLew 100 vector (Biebinger et al 1997). Transcription from the tetracyclineinducible procyclin promoter from this vector yields a transcript for an N terminus c-myc tagged TbPP5 protein.…”

“…TbPP5 RNAi cell lines of the T. brucei bloodstream and procyclic forms were generated using the stem-loop vector (Wang et al 2000) as described elsewhere (Anderson et al 2006). From these over-expressed and RNAi cells, Myctagged TbPP5 and TbPP5 double-stranded RNA were produced, respectively, upon induction with doxycycline (1.0 μg/ml).…”

“…Thus, for subsequent experiments, we heat shock cells at 40°C for 60 min. Cell suspension was evenly spread over poly-L-lysine (100 μg/ml in ddH 2 O) coated slides as described (Anderson et al 2006). Once the cells had settled, slides were washed with cold PBS to remove any unattached cells.…”

“…We have characterized PP5 from T. brucei previously (Chaudhuri 2001;Anderson et al 2006). T. brucei Hsp83 showed 70-72% homology with the mammalian Hsp90 and possesses a very high ATPase activity (Nadeau et al 1992).…”