# Protein Kinase Inhibitors and Oxidative Stress Modulate In Vivo Phosphorylation of Trypanosoma cruzi DNA Polymerase β

## TL;DR

The study explores how protein kinase inhibitors and oxidative stress affect the phosphorylation of a key enzyme in the parasite causing Chagas disease.

## Contribution

The study identifies dual protein kinase activity in Trypanosoma cruzi and its impact on DNA polymerase beta phosphorylation.

## Key findings

- Protein kinase inhibitors reduced TcPolβ Ser/Thr phosphorylation by about 50%.
- Genistein inhibited both Ser/Thr and Tyr phosphorylation in T. cruzi.
- Oxidative stress increased Tyr phosphorylation of TcPolβ and other proteins.

## Abstract

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.