Identification of novel serine/threonine protein phosphatases in Trypanosoma cruzi: a potential role in control of cytokinesis and morphology

We cloned two novel Trypanosoma cruzi proteins by using degenerate oligonuc leotide primers prepared against conserved domains in mammalian serine/thre onine protein phosphatases 1, 2A, and 2B. The isolated genes encoded protei ns of 323 and 330 amino acids, respectively, that were more homologous to t he catalytic subunit of human protein phosphatase 1 than to those of human protein phosphatase 2A or 2B, The proteins encoded by these genes have been tentatively designated TcPP1 alpha and TcPP1 beta. Northern blot analysis revealed the presence of a major 2.3-kb mRNA transcript hybridizing to each gene in both the epimastigote and metacyclic trypomastigote developmental stages, Southern blot analysis suggests that each protein phosphatase 1 gen e is present as a single copy in the T. cruzi genome. The complete coding r egion for TcPP1 beta was expressed in Escherichia coli by using a vector, p TACTAC, with the trp-lac hybrid promoter. The recombinant protein from the TcPP1 beta construct displayed phosphatase activity toward phosphorylase a, and this activity was preferentially inhibited by calyculin A (50% inhibit ory concentration [IC50], similar to 2 nM) over okadaic acid (IC50, similar to 100 nM). Calyculin A, but not okadaic acid, had profound effects on the in vitro replication and morphology of T. cruzi epimastigotes, Low concent rations of calyculin A (1 to 10 nM) caused growth arrest. Electron microsco pic studies of the calyculin A-treated epimastigotes revealed that the orga nisms underwent duplication of organelles, including the flagellum, kinetop last, and nucleus, but were incapable of completing cell division, At conce ntrations higher than 10 nM, or upon prolonged incubation at lower concentr ations, the epimastigotes lost their characteristic elongated spindle shape and had a more rounded morphology. Okadaic acid at concentrations up to 1 mu M did not result in growth arrest or morphological alterations to T. cru zi epimastigotes, Calyculin A, but not okadaic acid, was also a potent inhi bitor of the dephosphorylation of P-32-labeled phosphorylase a by T, cruzi epimastigotes and metacyclic trypomastigote extracts, These inhibitor studi es suggest that in T. cruzi, type 1 protein phosphatases are important for the completion of cell division and for the maintenance of cell shape.