“Trypanosoma cruzi CRKs and partners in the cell cycle”

TÉLLEZ-IÑÓN MA.TERESA

Glasgow, UK

Conferencia; COST ACTION B22 -DRUG DEVELOPMENT FOR PARASITIC DISEASES; 2006

University of Glasgow, UK

In Trypanosoma cruzi two CRKs genes TcCRK1 and TcCRK3 have been cloned. TcCRK1 levels and localization do not vary during the cell cycle. TzCRK1 is localized in the cytoplasm, discrete regions of the nucleus, and is highly concentrated in the kinetoplasto, suggesting a putative function in this organelle. TzCRK3 expression and activity was present throughout three life cycle stages of the parasite. Specific CDK inhibitors inhibited endogenous TzCRK3 activity and growth of epimastigotes as Flavopiridol. In synchronized epimastigotes with hydroxyurea, TzCRK3 activity peaked at G2/M boundary while the kinase associated to p13suc1-beads increased at the same time point remaining high until late G2/M. TzCRK3 expression was constant during the cell cycle showing the common pattern of CDK regulation. The results allow us to postulate that CRK3 shares functional homology with CDK1, and that it has a role controlling the G2/M transition in T. cruzi . The activity of CDKs is regulated by multiple regulatory mechanisms. At least four distinct posttranslational mechanisms positively regulated CDKs function, including binding of cyclins, binding of p13 (CKS) proteins phosphorylation and dephosphorylation of the protein by specific kinases and phosphatases. Searching proteins that regulate the cdc2-activity, we identified Tcp12CKS1 a member of the CKS family in the parasite Trypanosoma cruzi, similar to the p12 described previously in Leishmania (Mottram’s group). TcCKS1 is expressed in the three forms of T. cruzi. By using anti-Tcp12CKS1 antiserum, protein kinase activities were immunoprecipitated. The PK activity level varies depending on the stage analyzed, being lower in trypomastigotes and thus suggesting that different stages have different CKS-CRK complexes. Moreover, Flavopiridol, a known CDKs inhibitor, inhibited these PK activities. Immunoprecipitation and Western blot analyses demonstrated that in the epimastigote stage, p12CKS1 stably interacts with TcCRK1 and TcCRK3. In addition, Tcp12CKS1 was able to rescue the p13SUC1 null mutant of S. pombe. The functional complementation between the CKS proteins of two evolutionary distant organisms supports the role of Tcp12CKS1 as a key regulator in T. cruzi cell cycle.