Chagas disease is caused by the single-flagellated protozoan Trypanosoma cruzi, which affects several million people worldwide. Understanding the signal transduction pathways involved in this parasite’s growth, adaptation, and differentiation is crucial. Understanding the basic mechanisms of signal transduction in T. cruzi could help to develop new drugs to treat the disease caused by these protozoa. In the present work, we have demonstrated that Fetal Calf Serum (FCS) can quickly increase the levels of both phosphorylated and unphosphorylated forms of T. cruzi DNA polymerase beta (TcPolβ) in tissue-cultured trypomastigotes. The in vitro phosphorylation sites on TcPolβ by protein kinases TcCK1, TcCK2, TcAUK1, and TcPKC1 have been identified by Mass Spectrometry (MS) analysis and with antibodies against phosphor Ser-Thr-Tyr. MS analysis indicated that these protein kinases can phosphorylate Ser and Thr residues on several sites on TcPolβ. Unexpectedly, it was found that TcCK1 and TcPKC1 can phosphorylate a different Tyr residue on TcPolβ. By using a specific anti-phosphor Tyr monoclonal antibody, it was determined that TcCK1 can be in vitro autophosphorylated on Tyr residues. In vitro and in vivo studies showed that phorbol 12-myristate 13-acetate (PMA) can activate the PKC to stimulate the TcPolβ phosphorylation and enzymatic activity in T. cruzi epimastigotes.

Background/Objectives: Protein kinases play crucial roles in signal transduction pathways that regulate growth and differentiation in Trypanosoma cruzi. These protein kinases are attractive targets to develop new drugs to treat Chagas disease. Methods: We used several protein kinase inhibitors targeting the p38 MAPK, MEK, and ERK pathways to evaluate their effects on the in vivo phosphorylation status of T. cruzi proteins, particularly DNA polymerase beta (TcPolβ). We also used Genistein, a protein tyrosine kinase inhibitor, to assess its effects on global protein phosphorylation and TcPolβ phosphorylation. Also, we investigated the effect of oxidative stress on global tyrosine phosphorylation. Finally, we determined the phosphorylation sites on TcPolβ by the protein kinases TcPKC2 and TcWee570 in vitro. Results: p38 MAPK and MEK protein kinase inhibitors inhibited approximately 50% of the Ser/Thr phosphorylation of TcPolβ. Genistein inhibited both Ser/Thr and Tyr phosphorylation of several polypeptides in epimastigotes. Oxidative stress increases global Tyr phosphorylation by about twofold and also TcPolβ phosphorylation. TcPKC2 and TcWee570 were able to phosphorylate TcPolβ at both Ser/Thr and Tyr residues. Conclusions: Small-molecule protein kinase inhibitors can affect the phosphorylation status of TcPolβ in vivo. Since Genistein can inhibit both Ser/Thr and Tyr protein phosphorylation, and TcPKC2 and TcWee570 can phosphorylate both Ser/Thr and Tyr residues, it suggests the existence of dual protein kinases in T. cruzi. However, this possibility must be further studied.